Methods and compositions to alter the cell surface expression of phosphatidylserine and other clot-promoting plasma membrane phospholipids

ABSTRACT

A protein preparation that mediates Ca +2  transbilayer movement of phospholipid is disclosed. Additionally, a modified or mutated protein preparation, wherein the protein has a reduced ability to mediate transbilayer movement, is disclosed. In a preferred form of the invention, the protein has been modified such that post-translational modification can no longer occur.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 08/790,186, filedJan. 29, 1997, which claims priority to Ser. No. 60/015,385 filed Apr.02, 1996. Both of these applications are incorporated by reference as iffully set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

United States Government may have commercial rights under Grant R01HL36946 from Heart, Lung, & Blood Institute, National Institutes ofHealth.

BACKGROUND OF THE INVENTION

The exposure of phosphatidylserine (PS) and other aminophospholipids(aminoPL) on the surface of activated or injured blood cells andendothelium is thought to play a key role in the initiation andregulation of blood coagulation. De novo surface exposure ofaminophospholipids has also been implicated in the activation of bothcomplement and coagulation systems after tissue injury, and in removalof injured or apoptotic cells by the reticuloendothelial system.Although migration of these phospholipids (PL)from inner-to-outer plasmamembrane leaflets is known to be triggered by elevated intracellular[Ca²⁺] ([Ca²⁺]_(c)) and to be associated with vesicular blebbing of thecell surface, little is known about the cellular constituents thatparticipate in this process.

As described in Ser. No. 08/790,186, cell surface PS has a role incoagulation, programmed cell death and clearance by thereticuloendothelial system. Ser. No. 08/790,186 also describesregulation of the transmembrane distribution of PS, the role of calciumin the collapse of phospholipid asymmetry, and the role PL translocationin Scott Syndrome.

Bassè, et al. and Stout, et al. recently reported the purification andpreliminary characterization of an integral RBC membrane protein that,when reconstituted in liposomes, mediates a Ca²⁺-dependent transbilayermovement of PL mimicking plasma membrane PL reorganization evoked uponelevation of [Ca²⁺]_(c) (F. Bassè, et al., J. Biol. Chem.271:17205-17210, 1996; J. G. Stout, et al., J. Clin. Invest.99:2232-2238, 1997). Evidence that a protein of similar function mustalso be present in platelets was recently reported by Comfurius, et al.(P. Comfurius, et al., Biochemistry 35:7631-7634, 1996).

Needed in the art is a method for modulating the activity ofphospholipid scramblase within a cell, organ or tissue in which onewishes either to reduce the potential for thrombosis, clot formation, orcell clearance (by decreasing cellular PL scramblase expression oractivity) or to promote hemostasis or cell clearance (by increasingcellular PL scramblase expression or activity).

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the creation and use of antithromboticand thrombostatic reagents that rely on the properties of a proteinpreparation that mediates Ca²⁺-dependent transbilayer movement ofmembrane phospholipids.

In one embodiment, the present invention is a preparation of a plasmamembrane phospholipid scramblase (“PL scramblase”). Preferably, theprotein is approximately 35-37 kD as measured on a 12.5%SDS-polyacrylamide gel under reducing conditions. In a most preferredform of this invention, the preparation is a human or a mouse PLscramblase.

In one preferred embodiment of the present invention, the PL scramblasecomprises SEQ ID NO:2, representing human PL scramblase, possibly withconservative or functionally equivalent substitutions.

In the most preferred embodiment of the present invention, the PLscramblase, preferably comprising SEQ ID NO:2, has been modified by theaction of mammalian cellular enzymes to covalently incorporatephosphorous at one or more Thr, Ser, or Tyr residues or a fatty acid,preferably palmitate, at a cysteine residue.

The present invention is also a preparation of a murine cell protein,wherein the protein is a plasma membrane phospholipid scramblase,preferably wherein the protein is approximately 35 kD as measured on a12.5% SDS-polyacrylamide gel under reducing conditions.

In one preferred embodiment of the present invention, the murine PLscramblase comprises SEQ ID NO:4, possibly with conservative orfunctionally equivalent substitutions.

In the most preferred embodiment of the present invention, the murine PLscramblase comprising SEQ ID NO:4 has been modified by the action ofmammalian cellular enzymes to covalently incorporate phosphorous one ormore Thr, Ser, or Tyr residues, and a fatty acid, preferably palmitate,at a cysteine residue.

The present invention is also a DNA sequence encoding the PL scramblase.Preferably, this DNA sequence comprises SEQ ID NO:1. Most preferably,this DNA sequence comprises residues 211-1164 of SEQ ID NO:1.

The present invention is also a DNA sequence encoding the murine PLscramblase. Preferably, this DNA sequence comprises SEQ ID NO:3. Mostpreferably, the DNA sequence comprises residues 192-1112 of SEQ ID NO:3.

In another embodiment, the present invention is a method of preventingthe surface exposure of plasma membrane phospholipids and reducing theprocoagulant properties of a cell by delivering to the cell a mutantphospholipid scramblase. This scramblase is preferably mutated at a siteof post-translational modification, most preferably the site is selectedfrom the group consisting of Asp273-Asp284, Thr161 and Cys297 of humanPL scramblase SEQ ID NO:2 or the corresponding conserved residues inmouse or other mammalian PL scramblase.

In one embodiment, a gene construct encoding a mutant phospholipidscramblase is delivered to the cell. In an alternative embodiment, themutant protein itself is delivered.

The present invention is also an inhibitor of the PL scramblase activityof PL scramblase. This inhibitor may be an antisense nucleotide derivedfrom the DNA sequence of PL scramblase. In another embodiment, theinhibitor is a peptide sequence that is a competitive inhibitor of PLscramblase activity. In another embodiment, the inhibitor is anantibody, preferably a monoclonal antibody, raised against PLscramblase.

In another embodiment, the inhibitor works by modifying or inhibitingthe post-translational modifications of the PL scramblase that aredisclosed below in the Examples. For example, analysis of the primary PLscramblase sequence reveals a potential site of phosphorylation byprotein kinase C or other cellular kinase (Thr161), a potential site foracylation by fatty acid (Cys297), and a potential binding site for Ca²⁺ion provided by an EF-hand-like loop spanning residues Asp273-Asp284.These residues and motifs are conserved in the mouse PL scramblase.

In one embodiment, the inhibitor is a compound that preventsthioacylation of the protein.

In another embodiment, a mutant phospholipid scramblase is provided inwhich cysteine residues, preferably Cys297 of SEQ ID NO:2 (or theequivalent residue in the conserved region of another PL scramblase),have been replaced by alanine or other non-conservative substitution.

The present invention is also a method for preventing the surfaceexposure of plasma membrane phosphatidylserine, phosphatidylethanolamineand cardiolipin on the surface of in vitro stored leukocytes,lymphocytes, platelets or red blood cells. This method comprises thesteps of adding an inhibitor of PL scramblase activity to the storedblood cells.

The present invention is also a method for prolonging survival oftransplanted organs comprising the step of adding an inhibitor of PLscramblase activity to an organ perfusate during in vitro organ storage.The present invention is also a method for prolonging the survival oftransplanted cells, tissues, and organs by genetically engineering thecells to be transplanted so as to alter their expression of plasmamembrane PL scramblase in order to reduce exposure of PS and otherthrombogenic phospholipids at the plasma membrane surface, therebyreducing the risk of infarction due to fibrin clot formation.

The present invention is also a method for prolonging the in vivosurvival of circulating blood cells (erythrocyte, platelets, lymphocyte,PMN's, and monocytes) comprising the step of preventing surface exposureof plasma membrane phosphatidylserine on the surface of the cells byexposing the blood cells to an inhibitor of PL scramblase activity.

The present invention is also a method for preventing the procoagulantactivities of erythrocytes in sickle cell disease comprising the step ofinhibiting erythrocyte PL scramblase in a sickle cell patient.

The present invention is also a method for treating autoimmune andinflammatory diseases comprising the step of treating a patient with aninhibitor of the PL scramblase activity of PL scramblase.

The present invention is also a method for diagnosing individuals withreduced or elevated capacity for platelet-promoted orerythrocyte-promoted fibrin clot activity comprising the step ofquantifying the cellular expression of PL scramblase. This quantitationmay take the form of immunoblotting using an antibody to PL scramblase,an ELISA assay using an antibody to PL scramblase, flow cytometricanalysis of the binding of monoclonal antibody reactive against thepredicted extracellular domain of PL scramblase (residues Ser310-Tryp318of sequence disclosed in SEQ ID NO:2 or the equivalent residue in theconserved region of another PL scramblase) or using oligonucleotidesderived from PL scramblase cDNA and the polymerase chain reaction. Inone method of the present invention, the quantitation is performed byisolating PL scramblase from a patient blood sample, measuring theamount of PL scramblase isolated and comparing the measurement with acontrol sample. The measurement may be by isolating PL scramblase from apatient blood sample and measuring via densitometry the amount of PLscramblase protein electrophoresed in a stained electrophoretic gel.

It is an object of the present invention to provide a preparation of aPL scramblase.

It is another object of the present invention to genetically alter thelevel of expression of PL scramblase by delivery of cDNA representingsense or antisense nucleotide sequence ligated into a suitable mammalianexpression vector.

It is another object of the present invention to provide an inhibitor ofPL scramblase PL scramblase activity.

It is another object of the present invention to provide anantithrombotic agent.

It is another object of the present invention to create cells, tissue,and organs for transplantation that have increased potential forsurvival and reduced potential for causing fibrin clot formation andvascular thrombosis when grafted into a recipient host.

It is another object of the present invention to create an animal,preferably a mouse or pig, that has been genetically engineered so thatthe PL scramblase gene is not expressed.

Other objects, advantages and features of the present invention willbecome apparent after one of skill in the art reviews the specification,claims and drawings herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B is a comparison of the cDNA and deduced amino acidsequence of human PL scramblase (SEQ ID NOs:1 and 2).

FIGS. 1C and 1D is a comparison of the cDNA and deduced amino acidsequence of murine PL scramblase (SEQ ID NOs:3 and 4).

FIG. 2 is a bar graph illustrating immunoprecipitation of erythrocyte PLscramblase.

FIG. 3 is a graph of an activity assay of recombinant PL scramblase.

FIG. 4 is an immunoblot of PL scramblase in human erythrocytes andplatelets.

FIGS. 5A and 5B is a comparison of protein sequences of mouse and humanPL scramblase (SEQ ID NOs:2 and 4).

FIG. 6 is a bar graph of PL scramblase activity as a function ofmutational analysis of a putative EF hand loop motif contained in humanPL scramblase.

FIG. 7 graphs the Ca²⁺ dependence of mutant human PL scramblase.

FIG. 8 is a Western blot analysis of PL scramblase protein andcorresponding functional assay of PL scramblase activity in varioushuman cell lines.

FIG. 9A and B are fluorescence micrographs of GFP-PL scramblase intransformed Raji cells. FIG. 9A depicts fluorescence of cells expressingGFP; FIG. 9B depicts cells transfected with pEGFP-C2-PL scramblaseplasmid and expressing GFP-PL scramblase fusion protein.

FIG. 10 is a graph showing that the level of expression of PL scramblasedetermines plasma membrane sensitivity to intracellular calcium.

FIG. 11 is a bar graph illustrating inactivation of PL scramblase byhydroxylamine.

DETAILED DESCRIPTION OF THE INVENTION

In the description of the invention presented herein, Applicantsspecifically refer to numerical residue positions in both the PLscramblase amino acid and nucleotide sequence. These reference numbersrefer to the residue position in the amino acid or nucleotide sequencelisted in the Sequence Listing. When Applicants use these referencenumbers to describe a proposed mutated PL scramblase or nucleic acid,Applicants mean for these reference numbers to refer to the comparableor equivalent residue in that protein or amino acid. For example (seeFIGS. 5A and 5B), threonine 161 in the human protein sequence (SEQ IDNO:2) is equivalent to threonine 159 in the mouse protein sequence (SEQID NO:4). Although these residues have different reference numbers, theyare equivalent residues in the two sequences based upon conservedhomology in the aligned sequences. One may determine what a “equivalentresidue” is in an unknown PL scramblase sequence by deducing the highestprobability alignment to the human sequence using BLAST, FASTA or othersequence alignment tool commonly known to those skilled in the art.

1. Protein Preparations and Nucleic Acid Sequences

The Examples below disclose the purification and preliminarycharacterization of an integral RBC membrane protein that, whenreconstituted in liposomes, mediates a Ca²⁺-dependent transbilayermovement of PL mimicking plasma membrane PL reorganization evoked uponelevation of [Ca²⁺]_(c). Based on internal peptide sequence obtainedfrom the purified erythrocyte PL scramblase protein, we cloned the cDNA(SEQ ID NO:1) encoding this protein from a human K-562 erythroleukemiclibrary (Q. Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997). Thededuced human PL scramblase protein (SEQ ID NO:2) is a single chainpolypeptide of 318 amino acids with molecular weight of 35.1 kD andcalculated isoelectric point of 4.9. It is predicted to be a type 2membrane protein with a single transmembrane domain near the carboxylterminus (residues Ala291-Gly309), a short exoplasmic carboxyl terminalpeptide (residues Ser310-Trp318) with the remaining polypeptide(residues Met1-Lys290) in the cytosol.

The present invention involves the purification and characterization ofthis approximately 35-37 kD membrane protein that promotes aCa²⁺-dependent transbilayer redistribution of membrane phospholipidsincluding PS and PC, with properties similar to the PL scramblaseactivity that is evoked upon elevation of Ca²⁺ in the cytosol oferythrocytes and other cells. We have named this membrane protein “P37.”We mean for “P37” to be synonymous with “phospholipid scramblase” or “PLscramblase” and refer to these names interchangeably throughout thetext. By “phospholipid scramblase” or “PL scramblase activity,” we meanthe Ca²⁺ dependent transbilayer movement of plasma membranephospholipid.

In one embodiment, the present invention is a protein preparation of PLscramblase. In preferred embodiments of the present invention, thepreparation is of either human or mouse PL scramblase.

If one desires the human PL scramblase, preferably, the proteincomprises residues 1-318 of SEQ ID NO:2. More preferably, the PLscramblase comprises residues 85-307 of SEQ ID NO:2, representing onlythe most highly conserved residues of the human PL scramblase whenaligned against murine PL Scramblase (see FIGS. 5A and 5B).

If one desires the mouse PL scramblase, preferably, the proteincomprises residues 1-307 of SEQ ID NO:4. More preferably, the PLscramblase comprises residues 83-307 of SEQ ID NO:4, representing onlythe most highly conserved residues of the mouse PL scramblase whenaligned against human PL Scramblase (see FIGS. 5A and 5B).

In another embodiment, the protein comprises conservative substitutionsor functionally equivalent residues of the residues described in theparagraph above. By “functionally equivalent” we mean that theequivalent residues do not inhibit or disrupt the activity of the PLscramblase preparation. A protein with “equivalent” substitutions wouldhave at least a 10%, preferably 50%, activity of a native PL scramblasepreparation.

The Examples below demonstrate one method of isolating PL scramblasefrom human erythrocytes. After examination of the specification below,other methods of protein isolation will become apparent to one of skillin the art. The Examples below also describe an assay for themeasurement of PL scramblase activity. A suitable preparation of thepresent invention would have a PL scramblase activity of at least 10%that of the preparation described below in the Examples. Preferably, theactivity would be at least 50% that of the Examples described below.

We specifically envision that one may wish to isolate the PL scramblasefrom a variety of mammalian sources including human, mouse, pig, orother mammal.

In one embodiment of the invention, the PL scramblase is isolated fromerythrocyte membranes. In another embodiment, the protein is isolatedfrom one or more body tissues including, spleen, skin, lung or otherorgan. In another embodiment, the protein is produced by bacteria cells,such as E. coli cells, insect cells, or yeast, preferably in vitrocultures that are transfected with plasmid or viral vectors containingcDNA sequences identified at SEQ ID NOs:1 or 3 in the correct readingframe. The vector can be chosen from among protein expression vectorsknown to those skilled in the art. Preferable viral vectors includeretrovirus, adenovirus, and baculovirus vectors.

The present invention is also a recombinant DNA sequence encoding PLscramblase. A preferable DNA sequence encoding PL scramblase wouldcomprise the residues of SEQ ID NO:1 (human sequence) or SEQ ID NO:3(mouse sequence). A more preferable DNA sequence encoding PL scramblasewould comprise the nucleic acids 211-1164 of SEQ ID NO:1 or 192-1112 ofSEQ ID NO:3. The most preferred DNA sequence encoding PL scramblasewould comprise the nucleic acids 463-1137 of SEQ ID NO:1 or 438-1112 ofSEQ ID NO:3

One of skill in the art of molecular biology would know how to obtainother DNA sequences encoding the PL scramblase. For example, one mightsequence PL scramblase directly via standard protein sequencingtechniques. The peptide sequence could be analyzed to provideoligonucleotide probes for a human cDNA leukocyte library. (One suchcDNA library is available from Invitrogen in a pCDNA3 vector.)

By use of probes obtained from these and other the PL scramblases, onewould then be able to isolate other cDNA clones encoding the entire PLscramblase protein sequence from a species or cell culture of interest.SEQ ID NO:1 contains the entire open reading frame encoding human PLscramblase as well as flanking residues of 5′ and 3′ untranslatedsequence. The full-length translation of SEQ ID NO:1 is identified asSEQ ID NO:2. In the cDNA, this translated sequence would normally befollowed by the appropriate stop codon.

Based on the nucleotide sequence of human PL scramblase, the Examplesbelow disclose a full-length cDNA for murine PL scramblase from a mousefibroblast cDNA library (CLONETECH). The resulting cDNA (SEQ ID NO:3)predicts an open reading frame encoding a 307 residue polypeptide(molecular weight 33.9 kDa; calculated pI=4.9) (SEQ ID NO:4). Analysisof the murine PL scramblase protein revealed that it had virtually thesame apparent affinity for Ca²⁺ and the same activity in promotingtransbilayer movement of phospholipids as exhibited by the recombinanthuman protein.

Alignment of the murine and human PL scramblase proteins reveals 65%overall identity of sequence, with the most divergent sequence found inthe amino terminal portion of the protein. The murine carboxyl terminusis truncated, and does not include the predicted exoplasmic domain foundin the carboxyl terminus of human PL scramblase. This suggests thatresidues Ser310-Trp318 in human PL scramblase do not contribute to itsfunction. By contrast, segments of human PL scramblase polypeptide thatare implicated to participate in its phospholipid mobilizing function(detailed below), are highly-conserved in the mouse protein. Thesestructural motifs that are conserved in both human and mouse PLscramblase include: a single inside-outside transmembrane domain formembrane attachment (human residues Ala291-Gly309); a potential site ofphosphorylation by protein kinase C or other cellular kinase (Thr161 inhuman); a potential site of thiol-acylation with palmitic acid (Cys297in human); and a potential binding site for Ca²⁺ ion (residuesAsp273-Asp284 in human). Based on the best fit alignment of the humanand mouse protein sequences reported as SEQ ID NO:2 and SEQ ID NO:4, wededuce that the highly-conserved portions of the polypeptide,representing residues 85-309 of SEQ ID NO:2 of human PL scramblase andresidues 83-307 of SEQ ID NO:4 (i.e., the equivalent residues of mousePL scramblase) contains the portion of the protein required for PLscramblase activity.

The present invention is also a preparation of a modified or mutated PLscramblase wherein the PL scramblase has a reduced ability to mediatetransbilayer movement of lipids. By “reduced activity” we mean that themodified scramblase has less than 10% of the activity of the wild-typescramblase. Preferably, this activity is measured by the Ca²⁺ dependentmovement of fluorescent phospholipids in reconstituted proteoliposomes(see Examples 1 and 2). More preferably, this activity is measured bythe intracellular Ca²⁺-dependent movement of PS to the cell surface incells treated or transfected so as to express a modified or mutated PLscramblase (see Example 3).

Preferably, the protein is modified such that it is no longerpost-translationally modified, as described above. Most preferably, themodification occurs at amino acid residue Thr161, Cys297, orAsp273-Asp284.

The present invention is also a recombinant nucleic acid encoding amodified or mutated scramblase.

2. Modulators of PL Scramblase Activity.

The present invention is also a modulator, either an inhibitor orenhancer, of the PL scramblase activity of PL scramblase. Theinformation below in the Examples demonstrates a new understanding ofthe post-translational modification of PL scramblase that may be used todesign methods of modulating PL scramblase activity and mutated PLscramblases with modified scramblase activity. For example, analysis ofthe primary PL scramblase sequence reveals a potential site ofphosphorylation by protein kinase C or other cellular kinase (Thr161), apotential site for acylation by fatty acid (Cys297), and a potentialbinding site for Ca²⁺ ion provided by an EF-hand-like loop spanningresidues Asp273-Asp284. Knowledge of these post-translationalmodifications allows one to design specific inhibitors or modulators ofthe PL scramblase activity.

Therefore, as elaborated below in sections A, B and C, the presentinvention is a method of modulating PL scramblase activity by disruptingspecific post-translational modifications. In one embodiment, the methodcomprises exposing a PL scramblase molecule to a post-translationalmodification inhibitor and, thus, reducing PL scramblase activity. Thismethod will be useful in a variety of applications where reduction ofthe rate of clearance of a cell from the body or a reduction in clotpromoting and procoagulant activities of a cell is desired. Among thepost translational modification predicted to alter the activity of PLscramblase include insertion of the protein into phospholipid membranes,phosphorylation of the polypeptide by an intracellular protein kinase atone or more Tyr, Thr, or Ser residues, the addition of palmitate orother fatty acid by thioacylation through formation of a thioester bondat one or more Cys residues in the cytoplasmic or transmembrane domainsof the protein, the binding of one or more metal ions to the protein,the aggregation of the protein with itself or one or more cofactors,proteolytic degradation of the protein by one or more cytoplasmicproteases including by example calpains or caspases.

In another embodiment, the method comprises creating a gene constructencoding a modified PL scramblase. The scramblase will be modified atthe site of the post-translational modification described above. Thismodified gene may be used to transfect cells that one wishes to displaya reduction of clot promoting or procoagulant activities or to prolongthe survival of the cell in the body.

Sections A, B and C below describe specific residues that one may wishto mutate. One of skill in the art would be aware of general molecularbiological techniques that would enable one to acquire a PL scramblasegene, create the appropriate mutation, create the appropriate geneticconstruct, and transform the desired cell line.

In another embodiment, the inhibitor is an antisense nucleotide derivedfrom the DNA sequence encoding PL scramblase. One of skill in the artwould know how to create such an antisense nucleotide from the cDNAsequence of PL scramblase.

In another embodiment, the inhibitor is an antibody, preferably amonoclonal antibody, raised against PL scramblase. One of skill in theart would know how to make an antibody preparations from the purifiedprotein preparation described below.

A. Cysteine thioester

Our discovery of the presence of a conserved site for Cysthiol-acylation in the transmembrane domains of human and mouse PLscramblase (Cys297 in human) suggests that PL scramblase polypeptide ispost-translationally modified by the attachment of fatty acid, and thatthis thiol-acylation is required for normal expression of its biologicalactivity. Attachment of fatty acid (predominantly palmitic acid) byacylation of the cysteinyl thiol residue has been shown to regulate thebiological activity of a variety of cellular proteins (G. Milligan, etal., Trends Biochem. Sci. 20:181-185, 1995; M. J. Schlesinger, et al.,In: Lipid Modification of Proteins, pp. 1-19, CRC Press, Boca Raton,Fla., 1993).

One embodiment of the present invention is a method to prevent egress ofPS and other clot-promoting and procoagulant phospholipids to cellsurfaces by preventing or reversing the acylation of cysteines in plasmamembrane PL scramblase protein. In one embodiment of the presentinvention, one would create a mutated scramblase, wherein cysteine 297(or the equivalent residue in the conserved region of another PLscramblase) is no longer available for post-translational modification.This may be by substituting the cysteine with a alanine, serine oranother non-functionally equivalent amino acid residue. The cDNAencoding this mutated scramblase may be placed in a vector expressionsystem and used to transfect cells of interest. We envision that themutated scramblase will out-compete native scramblase and, thus, reducescramblase activity.

In another embodiment, the method preferably comprises the steps ofexposing a PL scramblase to a thiolacylation inhibitor and inhibiting PLscramblase activity. Applicants note that the thiolacylation inhibitorcould either inhibit thiolacylation directly, block the site ofthiolacylation, or hydrolyze pre-existing thioester-linked fatty acidsattached to the protein. Applicants specifically envision that thethiolacylation may be prevented by compounds selected from the class ofspecific antibodies against the protein that react at the site ofthioacylation, thiol-reactive compounds that covalently modify cysteineresidues (including by example N-ethyl maleimide, iodoacetamide, orpyridyldithioethylamine), an inhibitor of enzyme acyltransferasesincluding by example an esterase inhibitor chosen from among carbamates(e.g., physostigmine)and organophosphorus (e.g.,diisopropylfluorophosphate) compounds that are reactive at the activesite of such enzymes. Such a method will be useful for many of theobjects described above, such as treating cells, tissues, and organs fortransplantation to reduce potential for causing fiber and clot formationin vascular thrombosis when grafted into a recipient host. The methodcould also be used to provide an antithrombotic therapeutic effect. Themethod could also be used to increase in vivo survival of the transfusedor transplanted cell by suppressing exposure of PS or otheraminophospholipids at the cell surface.

B. Peptide residues involved in binding Ca²⁺.

The phospholipid transport function of PL scramblase is activated byCa²⁺ with apparent affinity of 50-100 micromolar, implying a relativelylow affinity binding site for the calcium ion within the polypeptide (Q.Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997; J. G. Stout, et al.,J. Clin. Invest. 99:2232-2238, 1997; F. Bassè, et al., J. Biol. Chem.271:17205-17210, 1996). The deduced protein sequence of mouse and humanPL scramblase reveals an extensive segment of highly conserved sequenceextending through residue Glu306 (or the equivalent residue in theconserved region of another PL scramblase). The predicted secondarystructure through this portion of the protein reveals that it containstwo short alpha-helical segments near the C-terminus that are separatedby a 12-residue acidic loop. In both proteins (human and mouse), theC-terminal alpha helix represents a predicted transmembrane segment witha strongly-preferred inside-to-outside orientation, whereas sequencecontained within the adjacent 12-residue acidic loop conforms in-part toa consensus sequence that is characteristic of an EF-hand Ca²⁺-bindingmotif (S. Nakayama, et al., Annu. Rev. Biophys. Biomol. Struct.23:473-507, 1994). In this motif, residues in positions 1, 3, 5, 7, 9and 12 contribute to octahedral coordination of the Ca²⁺ ion, with theresidues in position 1 [Asp], 3 [Asp, Asn, or Ser] and 12 [Asp or Glu]being those most highly conserved. In order to gain insight into whetherthis segment of the protein might be directly involved in theCa²⁺-dependent reorganization of membrane PL mediated by PL scramblase,we expressed mutant human PL scramblase with Asp→Ala substitutions atpositions corresponding to residues 1 (i.e., Asp273), 3 (i.e., Asp275),and 12 (i.e., Asp284) of this putative 12 residue EF-hand loop. Whereasmutations in positions 1 or 12 lead to a partial loss of function,mutation in position 3 resulted in complete inactivation of theCa²⁺-dependent response. The partial loss in activity of PL scramblasewith mutations in positions 1, 3, or 12 was accompanied by a significantreduction in apparent avidity for Ca²⁺. These data identify the segmentof human PL scramblase between Asp273-Asp284 as containing the essentialbinding site for Ca²⁺ and suggest that the activity of this protein canbe selectively inhibited by blocking access of Ca²⁺ to this segment ofthe polypeptide or by modifying residues contained in this segment ofthe polypeptide.

Therefore, in another embodiment, the present invention is a method forinhibiting the clot-promoting and procoagulant activity of the plasmamembrane by preventing the binding of intracellular Ca²⁺ to the PLscramblase polypeptide. In one embodiment, the method comprisesconstructing a mutant PL scramblase, wherein the PL scramblase ismutated between Asp273 and Asp284 so that the scramblase does not bindCa²⁺ with the same affinity as PL scramblase. This mutant gene may beused, by methods known to one of skill in the art, to transfect a cellor cell line in which one wishes to modulate the clot promoting orprocoagulant properties. We envision that the mutated PL scramblase willout-compete native PL scramblase and thus modulate, preferably reduce,PL scramblase activity in the cell or cell line.

In another embodiment, this method preferably comprises the steps ofexposing a PL scramblase to a calcium binding inhibitor and reducing PLscramblase activity. Applicants specifically envision that thisinhibitor may be either a direct inhibitor of calcium binding or wouldbind to the residues described above and block calcium binding. Thismethod, as above, would be useful to prepare cells, tissues and organsfor transplantation and as a therapeutic antithrombotic.

C. Modification of PL Scramblase Through Phosphorylation by CellularProtein Kinases

As another embodiment, the present invention includes a method toprevent phosphorylation of PL scramblase at one or more Tyr, Thr or Serresidues by inhibiting the action of intracellular Tyr or Ser/Thrkinases. The amino acid sequence of PL scramblase reveals potentialsites of phosphorylation at Tyr, Thr, or Ser residues by cellularprotein kinases, including the conserved motif Thr161-Leu162-Arg163 ofhuman SEQ ID NO:2 (corresponding to Thr159-Leu160-Arg161 of mouse SEQ IDNO:4) predicting phosphorylation by protein kinase C (Q. Zhou, et al.,supra, 1997 and FIG. 5). Protein phosphorylation by one or more cellularprotein kinases is known to regulate many aspects of cell function,which can include both the activation and inactivation of specificenzyme activities. In the specific case of PL scramblase, depletion ofcellular ATP has been shown to inhibit surface exposure ofphosphatidylserine on erythrocytes exposed to elevated [Ca²⁺]_(c) (D. W.Martin, et al., J. Biol. Chem. 270:10468-10474, 1995). In combinationwith our discovery of conserved motifs for phosphorylation of PLscramblase, we propose that normal PL scramblase activity requiresconstitutive phosphorylation of the polypeptide. Such phosphorylationsinvariable occur at one or more tyrosines (ie., by tyrosine proteinkinases) or at one or more serines or threonines (i.e. by Ser/Thrprotein kinases). The specific sites of phosphorylation within a givenprotein are readily identified by finding conserved sequence motifspredictive of phosphorylation, and confirmed using methods known tothose skilled in the art. Such methods include metabolically labelingthe cellular proteins with ³²P, purifying the protein using specificantibody, and identifying the specific residues of polypeptide sequencethat contain covalently bound ³²P, standardly performed by trypticcleavage of the isolated protein, HPLC separation of resulting peptides,and identification of the phosphorylated residues by either Edmandegradation or mass spectroscopic analysis.

As another embodiment, the Examples below specifically disclose Thr161(or the equivalent residue in the conserved region of another PLscramblase) as a single predicted site of phosphorylation by proteinkinase C. Disruption of this phosphorylation or modification of theparticular residues involved would modify PL scramblase activity.Therefore, the present invention is a method for altering theprocoagulant activity of the plasma membrane by preventingphosphorylation by protein kinases, such as protein kinase C. In oneembodiment, the method comprises creating a mutant PL scramblase,wherein the mutant PL scramblase does not contain a site capable ofphosphorylation by cellular protein kinase. Preferably, the mutant PLscramblase is mutated at Thr161. One then creates a gene constructcapable of expressing the mutated PL scramblase and transfects a cell orcell line of interest. In this manner, one introduces a mutant PLscramblase into the cell or cell line and out competes native orwild-type PL scramblase, thus altering the PL scramblase activity of thecell. In another embodiment, the method preferably comprises the stepsof exposing a PL scramblase to a phosphorylation inhibitor and thusaltering PL scramblase activity. Applicants specifically envision thatthis inhibitor may be either a general phosphorylation inhibitor or mayspecifically block phosphorylation at Thr161. As described above, themethod would be useful to prepare cells tissues and organs fortransplantation and as a therapeutic antithrombotic.

3. Expression of PC Scramblase in Human Platelet, Human Endothelium andother Cell Types

Our results described below in the Examples confirm that the level ofexpression of plasma membrane PL scramblase can determine the extent towhich PS is mobilized to the cell surface upon elevation of [Ca²⁺]_(c),and suggest that this protein normally functions to mediate theredistribution of plasma membrane phospholipids in response to the entryof calcium into the cytosol.

These data provide the first experimental demonstration that thecellular potential to mobilize PS and other procoagulantaminophospolipids from plasma membrane inner leaflet to the cellsurface—and thereby expose binding sites for factor Va or other plasmacoagulation factor—can be manipulated by selectively altering the levelof expression of a particular cellular protein, either through directtransfection with the PL scramblase cDNA, by another interventionaffecting either total cellular expression of PL scramblase protein or apost-translational modification of the PL scramblase polypeptide that isessential for its PS mobilizing function in the plasma membrane.

In one embodiment, the present invention is a method of eitherincreasing or decreasing the clot-promoting and procoagulant propertiesof cell surfaces by either increasing or decreasing the level ofcellular expression PL scramblase mRNA and protein.

4. Other Embodiments

The present invention is also a method for preventing the surfaceexposure of plasma membrane phospholipids, such as phosphatidylserine,phosphatidylethanolamine and cardiolipin, on the surface of in vitrostored blood cells (including, platelets, red blood cells, lymphocytes,or leukocytes) by adding an inhibitor or modulator of the PL scramblaseactivity of PL scramblase to the stored cells.

The present invention is also a method for prolonging survival oftransplanted organs and grafts comprising the step of adding aninhibitor of PL scramblase PL scramblase activity to an organ perfusateduring in vitro organ storage. The present invention is also a methodfor prolonging the survival of transplanted cells, tissues, and organsby genetically engineering the cells to be transplanted so as to altertheir expression of plasma membrane PL scramblase in order to reduceexposure of PS and other thrombogenic phospholipids at the plasmamembrane surface, thereby reducing the risk of infarction due to fibrinclot formation.

Therefore, in one embodiment, the present invention is a geneticallyengineered cell for transplantation into a human or animal wherein thecell has a lowered PL scramblase expression. Preferably, the cellexpresses no PL scramblase. Preferably, this cell comprises a nucleotidemolecule which is expressed by the cell and which codes for proteininhibiting the activity of PL scramblase. In another preferableembodiment, the promotor of the PL scramblase gene is altered to eitherincrease or decrease the expression of the gene. One of skill in the artof molecular biology would envision methods to create these alteredcells.

The present invention is also an animal, such as a mouse or pig, thathas been genetically manipulated to “knock out” PL scramblaseexpression. Such an animal may be created by many variations oftechniques known to one of skill in the art. Most preferably, one woulddelete by homologous recombination one or more exons of the PLscramblase gene within the chromosomal DNA of the appropriate embryonicstem cell of mouse, pig, or other animal. In the mouse, those exons mostfavored for deletion by homologous recombination are those that includepart or all of DNA sequence between residues 438-1112 of SEQ ID NO:3,representing the conserved portion of the cDNA open reading framerequired for expression of functional PL scramblase protein. Thoseembryonic stem cells showing the PL scramblase gene deletion aresurgically implanted within the uterus at the appropriate time in theestrus cycle. The resulting animals carrying the defective gene are thenbred to homozygosity for the PL scramblase gene deletion defect.

Preferably, the engineered cell is selected from the group consisting ofendothelial cells, fibroblasts, epithelial cells, skeletal cells,cardiac and smooth muscle cells, hepatocytes, pancreatic islet cells,bone marrow cells, astrocytes, and Schwann cells. The present inventionis also a prosthesis for implantation in an animal or human having thegenetically engineered cells attached thereto. In one embodiment, theprosthesis is a vascular graft.

The present invention is also a method for prolonging the in vivosurvival of circulating blood cells comprising the step of preventingsurface exposure of plasma membrane phosphatidylserine on thecirculating blood cells by inhibiting the function of plasma membrane PLscramblase. One may also wish to prevent the procoagulant properties oferythrocytes in sickle cell disease by inhibiting erythrocyte PLscramblase in a sickle cell patient.

The present invention is also a method for treating autoimmune andinflammatory diseases, such as disseminated intravascular coagulation,vascular thrombosis, fibrin generation during cardiopulmonary bypassprocedures, rheumatoid arthritis, systemic lupus erythematosus,thrombotic thrombocytopenic purpura, heparin-associated thrombosis, andorgan transplant rejection comprising the step of treating a patientwith an inhibitor of the PL scramblase activity PL scramblase.

The present invention is also a method for diagnosing individuals withreduced or elevated capacity for platelet-promoted orerythrocyte-promoted fibrin clot activity by quantitating the level ofcellular expression of PL scramblase in the individual. This method maybe performed by using an antibody to PL scramblase in an immunoblot,ELISA, or fluorescence flow cytometric method. The method may also beperformed using oligonucleotides derived by PL scramblase cDNA in thepolymerase chain reaction. In another embodiment, the method may beperformed by isolating PL scramblase from a whole blood sample,measuring the amount of PL scramblase isolated and comparing themeasurement with a control sample.

One may wish to use the protein preparation of the present invention asa hemostatic agent by topically applying the protein preparation to awound area in a freely bleeding patient.

EXAMPLES

In the Examples below, we identify the cellular component that functionsto mediate the Ca²⁺-dependent reorganization of plasma membranephospholipids, we identify the essential structural elements of thisprotein that are required for its phospholipid transporting function,and we describe methods for inhibiting or accelerating egress of PS tothe surface of activated, injured, or apoptotic cells.

EXAMPLE 1 Purification, Sequencing and Molecular Cloning of Human PLScramblase

A. Summary

The rapid movement of phospholipids (PL) between plasma membraneleaflets is thought to play a key role in expression of plateletprocoagulant activity and in clearance of injured or apoptotic cells.U.S. Ser. No. 08/790,186, upon which this application claims priority,discloses isolation of a ˜37 kDa protein in erythrocyte membrane thatmediates Ca²⁺-dependent movement of PL between membrane leaflets,similar to that observed upon elevation of Ca²⁺, in the cytosol [F.Bassè, et al. J. Biol. Chem. 271:17205-17210, 1996]. Based on internalpeptide sequence obtained from this protein, a 1,445 bp cDNA was clonedfrom a K562 cDNA library. The deduced protein is a proline-rich, type IIplasma membrane protein with a single transmembrane segment near theC-terminus. Antibody against the deduced C-terminal peptide was found toprecipitate the ˜37 kDa red blood cell protein and absorb PL scramblaseactivity, confirming the identity of the cloned cDNA to erythrocyte PLscramblase. Ca²⁺-dependent PL scramblase activity was also demonstratedin recombinant protein expressed from plasmid containing the cDNA.Quantitative immunoblotting revealed an approximately 10-fold higherabundance of PL scramblase in platelet (˜10⁴ molecules per cell) than inerythrocyte (˜10³ molecules/cell), consistent with apparent increased PLscramblase activity of the platelet plasma membrane. PL scramblase mRNAwas found in a variety of hematologic and non-hematologic cells andtissues, suggesting that this protein functions in all cells.

B. Experimental Procedures

All experimental procedures and abbreviations are as set out in U.S.Ser. No. 08/790,186, unless otherwise noted. anti-306-318, affinitypurified rabbit antibody (IgG fraction) against peptide [C]ESTGSQEQKSGVW(SEQ ID NO:5); EST, expressed sequence tag; IPTG,isopropyl-β-D-thiogalactopyranoside; MBP, maltose binding protein;

Materials. Egg yolk phosphatidylcholine (PC), brain phosphatidylserine(PS), 1-palmitoyl 2-oleoyl phosphatidic acid,1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine(NBD-PC) and1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphoserine(NBD-PS) were obtained from Avanti Polar Lipids. Expressed sequence tag(EST) clone with GenBank accession number gb AA143025 was obtainedthrough American Type Culture Collection (ATCC962235). All restrictionenzymes and amylose resin were from New England BioLabs, Inc. Klentaqpolymerase was from Clontech Laboratories, wheat germ agglutininSepharose from Sigma, IPTG from Eastman Kodak, factor Xa fromHaematologic Technologies, and Bio-Beads SM-2 were from BioRad.N-octyl-β-D-glucopyranoside (OG) and Glu-Gly-Arg chloromethyl ketonewere from Calbiochem. Sodium dithionite (Na₂S₂O₄, Sigma) was freshlydissolved in 1 M Tris pH 10 at a concentration of 1 M.N-Octyl-β-D-glucopyranoside (OG) was purchased from Calbiochem. Sodiumdithionite (Na₂S₂O₄₁, Sigma) was freshly dissolved in 1 M Tris pH 10 ata concentration of 1 M.

PL Scramblase isolation. Human PL scramblase protein and cDNA wasobtained as described in Ser. No. 08/790,186.

Cloning of PL scramblase into pMAL-C2 expression vector. In order toexpress PL scramblase as a fusion protein with maltose binding protein(MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (New EnglandBioLabs). PCR was performed on a full-length clone using theprimers^(5′)TCA GAA TTC GGA TCC ATG GAC AAA CAA AAC TCA CAG ATG^(3′)(SEQID NO:6) with an EcoR1 site before the ATG start codon and ^(5′)GCT TGCCTG CAG GTC GAC CTA CCA CAC TCC TGA TTT TTG TTC C³ (SEQ ID NO:7) with aSalI site after the stop codon. KlenTaq polymerase (Clontech) was usedto ensure high fidelity amplification. The PCR product was digested withEcoR1 and SalI and isolated by electrophoresis on 1% low melting agarosegel and purification with Wizard kit (Promega). The amplified cDNA wascloned into pMAL-C2 vector digested with EcoRI and SalI, immediately 3′of MBP. This construct was amplified in E. coli strain TB1, and sequenceof the cDNA insert of plasmids from single colonies confirmed.

Expression and purification of PL scramblase-MBP fusion protein. Ten mlof E. coli TB1 transformed with PL scramblase cDNA-pMAL-C2 were used toinoculate 1 L of rich LB containing 2 mg/ml glucose, 100 μg/mlampicillin, and the bacteria were allowed to grow for about 4 hours at37° C. When A₆₀₀ reached ˜0.5, IPTG was added to a final concentrationof 0-3 mM. After 2 hours of incubation at 37° C., the cells werecentrifuged at 4000×g for 20 minutes. The cell pellet was suspended in50 ml of 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF (columnbuffer), and subjected to a freeze/thaw cycle. After sonication (3×30seconds on ice) and centrifugation at 43,000×g for 1 hour, thesupernatant was applied to 10 ml of amylose resin. The column was washedwith 20 volumes of column buffer, and the scramblase-MBP fusion proteineluted with the same buffer containing 10 mM maltose. Digestion ofMBP-PL scramblase protein with factor Xa was routinely performed at{fraction (1/100)} (w/w) ratio of enzyme and monitored by SDS-PAGE. Inaddition to MBP, the product of this digest is the PL scramblasetranslation product containing the N-terminal extensionIle-Ser-Glu-Phe-Gly-Phe (codons −6 to −1).

Reconstitution of PL scramblase or scramblase fusion protein intoproteoliposomes. Reconstitution and functional activity were performedessentially as previously described (F. Bassè, et al., J. Biol. Chem.271:17205-17210, 1996; J. G. Stout, et al., J. Clin. Invest.99:2232-2238, 1997). Briefly, a mixture of PC and PS (9:1 molar ratio)was dried under a stream of nitrogen and resuspended in 100 mM Tris, 100mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer). Protein samples to bereconstituted were added to the liposomes at a final lipid concentrationof 4 mg/ml in the presence of 60 mM OG, and dialyzed overnight at 4° C.against 200 vol of Tris buffer containing 1 g/L Bio-Beads SM-2. Toliberate PL scramblase from MBP, the proteoliposomes were incubated 3 hat room temperature in the presence of {fraction (1/40)} (w/w) factorXa. The digestion was terminated by addition of 100 μM Glu-Gly-Argchloromethyl ketone. Completeness of the digest was monitored bySDS-PAGE. Following dialysis, the proteoliposomes were labeled in theouter leaflet by addition of 0.25 mol % fluorescent NBD-PC (in dimethylsulfoxide, final solvent concentration 0.25%).

PL Scramblase activity. Scramblase activity was measured as previouslydescribed (F. Bassè, et al., supra, 1996; J. G. Stout, et al., supra,1997 and in U.S. Ser. No. 08/790,186). Routinely, proteoliposomeslabeled with NBD-PC were incubated for 2 hours at 37° C. in Tris bufferin the presence or absence of 2 mM CaCl₂. Proteoliposomes were diluted25-fold in Tris buffer containing 4 mM EGTA and transferred to a stirredfluorescence cuvet at 23° C. Initial fluorescence was recorded (SLMAminco 8000 spectrofluorimeter; excitation at 470 nm, emission at 532nm), 20 mM dithionite was added, and the fluorescence continuouslymonitored for a total of 120 seconds. The difference in non-quenchablefluorescence observed in presence vs. absence of CaCl₂ was attributed toCa²⁺-induced change in NBD-PC located in the outer leaflet (F. Bassè, etal., supra, 1996; J. G. Stout, et al., supra, 1997; J. C. McIntyre andR. G. Sleight, Biochemistry 30:11819-11827, 1991). Ionized [Ca²⁺] wascalculated using FreeCal version 4.0 software (generously provided byDr. Lawrence F. Brass, University of Pennsylvania, Philadelphia, Pa.).

Antibody against PL Scramblase C-terminal peptide. The peptideCESTGSQEQKSGVW (SEQ ID NO:5), corresponding to amino acids 306-318 ofthe predicted open reading frame of PL scramblase with an addedN-terminal cysteine, was synthesized and conjugated to keyhole limpethemocyanin (Protein Core Facility, Blood Research Institute). Antiserumto this protein was raised in rabbit (Cocalico Biologicals, Inc.) andthe IgG fraction isolated on Protein A Sepharose-CL4B (Sigma).Peptide-specific antibody was isolated by affinity chromatography onUltraLink Iodoacetyl beads (Pierce) to which peptide CESTGSQEQKSGVW (SEQID NO:5) was conjugated. This affinity-purified antibody (anti-306-318)was used for immunoprecipitation and Western blotting of PL scramblase(below) Immunoprecipitation of PL scramblase. PL scramblase purifiedfrom human erythrocytes was ¹²⁵I-labeled with Iodogen (Pierce), freeiodide removed by gel filtration, and the protein incubated (4° C.,overnight) with either anti-306-318, or an identical quantity ofpre-immune rabbit IgG (1 mg/ml in 150 mM NaCl, 10 mM MOPS, 50 mM OG, pH7.4) or no IgG as control. The IgG was precipitated with protein ASepharose, washed exhaustively, and protein bands resolved by 8-25%SDS-PAGE (Phast System, Pharmacia Biotech Inc.) under reducingconditions. Radioactive bands were visualized by autoradiography. Inorder to determine whether antibody to this peptide specifically removedthe functional activity associated with the purified erythrocyte PLscramblase protein, the supernatant fractions remaining afterimmunoprecipitation were reconstituted in liposomes for activitymeasurements, performed as described above. For these experiments,unlabeled erythrocyte PL scramblase substituted for the ¹²⁵I-labeledprotein.

Western Blot Analysis. 2×10⁸ washed platelets, 2×10⁸ erythrocyte ghostmembranes, 0.9 pmoles of purified recombinant PL scramblase (obtained byfactor Xa digest of the PL scramblase-MBP fusion protein), or 0.3 pmolesof PL scramblase purified from human erythrocyte were each denatured byboiling in 40 μl sample buffer containing 10% SDS, 4% β-mercaptoethanol,and 1 mM EDTA, and protein bands resolved by SDS-PAGE. After transfer tonitrocellulose, the blocked membrane was incubated with 1 μg/ml ofanti-306-318, and the blot developed with horseradishpreoxidase-conjugated goat anti-rabbit IgG (Sigma) usingChemiluminescence Reagent (Dupont).

Protein Concentrations. Protein concentrations were estimated based uponoptical density at 280 nm, using extinction coefficients (M⁻¹cm⁻¹) of39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scramblase-MBPfusion). PL scramblase contained in human platelet and erythrocytemembranes was estimated by quantitative immunoblotting of the detergentextracts, with reference to known quantities of purified MBP-PLscramblase fusion protein.

Northern Blot Analysis. Human multiple tissue northern blot and humancancer cell line multiple tissue northern blot membranes were obtainedfrom Clontech. The blots were prehybridized with ExpressHyb (Clontech)at 68° C. for 30 minutes and hybridized with ExpressHyb containing 5ng/ml ³²P-labeled PL scramblase cDNA probe at 68° C. for 1 hour, thenwashed and exposed to X-ray film. After development, the blots werestripped and hybridized with ³²P-labeled β-actin cDNA probe usingidentical conditions.

C. Results and Discussion

U.S. Ser. No. 08/790,186 describes the initial purification of PLscramblase from human erthyrocyte membrane and analysis of cyanogenbromide fragments. The fragments were used to obtain an entire PLscramblase DNA alone (FIGS. 1A, B, C and D).

FIGS. 1A and B illustrates the cDNA and deduced amino acid sequence ofhuman PL scramblase. The deduced amino acid sequence of the predictedopen reading frame is shown under the nucleotide sequence. The 32residues of peptide sequence that were obtained from cyanogen bromidedigest of purified erythrocyte PL scramblase are indicated by singleunderline. Also indicated are the residues comprising a predictedinside-to-outside transmembrane domain (Ala291-Gly309; double underline)and protein kinase C phosphorylation site (Thr 161; asterisk). SeeExperimental Procedures for details.

Analysis of the cDNA-derived protein sequence (Tmpred program, ISRECserver, Univ. of Lausanne, Epalinges, Switzerland) revealed astrongly-preferred (p<0.01) inside-to-outside orientation of thepredicted 19 residue transmembrane helix, consistent with a type IIplasma membrane protein. Most of the polypeptide (residues 1-290)thereby extends from the cytoplasmic membrane leaflet, leaving a shortexoplasmic tail (residues 310-318). The predicted orientation of thisprotein is consistent with the anticipated topology of PL scramblase inthe erythrocyte membrane, where lipid-mobilizing function is responsiveto [Ca²⁺] only at the endofacial surface of the membrane (P. Williamson,et al., Biochemistry 31:6355-6360, 1992; E. F. Smeets, et al., Biochim.Biophys. Acta Bio-Membr. 1195:281-286, 1994; F. Bassè, et al., supra,1996; J. G. Stout, et al., supra, 1997; P. Williamson, et al.,Biochemistry 34:10448-10455, 1995; D. L. Bratton, J. Biol. Chem.269:22517-22523, 1994).

In order to confirm that the cDNA we cloned from the K562 cDNA libraryactually encodes the same protein purified as PL scramblase from humanerythrocyte membrane, we raised a rabbit antibody against the deducedC-terminus predicted from the open reading frame of the cloned cDNA(codons 306-318).

FIG. 2 illustrates immunoprecipitation of erythrocyte PL scramblase. PLscramblase purified from human erythrocytes was precipitated with eitheranti-306-318 IgG (bar 1), or with pre-immune rabbit IgG (bar 2) andprotein remaining in the supernatant reconstituted into liposomes formeasurement of residual PL scramblase activity. Data normalized to PLscramblase activity measured for identical controls omitting antibody(100%; bar 3). Error bars denote mean ± SD (n=4).

As shown in FIG. 2, this antibody precipitated the ˜37 kDa red cellprotein we tentatively identified as PL scramblase, and also absorbedthe functional activity detected in this isolated erythrocyte membraneprotein fraction. We often observed the partial proteolysis of 37 kDa PLscramblase to a polypeptide of ˜30 kDa. The apparent susceptibility ofthis protein to proteolytic degradation may account for the reportedrapid loss of activity observed in earlier attempts to purify PLscramblase from platelet (P. Comfurius, et al., Biochemistry35:7631-7634, 1996).

Expression and membrane reconstitution of recombinant PL scramblase.Recombinant PL scramblase was expressed in E. coli as fusion proteinwith MBP, purified by amylose affinity chromatography, and incorporatedinto PC/PS liposomes for assay of PL scramblase activity. Whenincorporated into liposomes, the recombinant protein mediated aCa²⁺-dependent transbilayer movement of NBD-PC mimicking the activity ofPL scramblase isolated from erythrocyte. PL scramblase activity wasobserved both for the chimeric MBP-PL scramblase fusion protein, and forrecombinant PL scramblase liberated from MBP through proteolyticdigestion with factor Xa (FIG. 3).

FIG. 3 depicts an activity assay of recombinant PL scramblase. PurifiedPL scramblase-MBP fusion protein (0-43×10¹⁻¹ moles; abscissa) wasreconstituted into liposomes (1 μmole total PL) and MBP proteolyticallyremoved by incubation with factor Xa in presence of 0.1 mM EGTA. Afterdigest to release MBP, the proteoliposomes were recovered fordetermination of PL scramblase activity, measured in the absence (∘) orpresence () of 2 mM CaCl₂ as described in Experimental Procedures. Dataare corrected for non-specific transbilayer migration of NBD-PC probe inidentically-matched control liposomes containing either MBP or no addedprotein (<2% NBD-PC sequestered; not shown). Error bars denote mean ± SD(n=3). Data of single experiment, representative of two so performed.Similar results were also obtained for proteoliposomes containing intactPL scramblase-MBP fusion protein, omitting the factor Xa digest (notshown).

By contrast, no such activity was observed for control proteinconsisting of the pMAL-C2 translation product MBP lacking the PLscramblase cDNA insert. The specific PL mobilizing activity ofrecombinant PL scramblase expressed and purified from E. coli wasapproximately 50% of that observed for the endogenous protein purifiedfrom the erythrocyte membrane, which is likely due to incomplete foldingof the recombinant protein. Half-maximal [Ca²⁺] required for activationwas approximately 100-200 μM for recombinant protein purified from E.coli versus ˜40 μM for the erythrocyte-derived protein, raising thepossibility that altered folding or an unknown post-translationalmodification in mammalian cells affects the putative Ca²⁺ binding site(F. Bassè, et al., supra, 1996; J. G. Stout, et al., supra, 1997). Inaddition to activation by Ca²⁺, the transbilayer migration of PL inerythrocytes is accelerated upon acidification of the inside leaflet topH<6.0 (in absence of Ca²⁺), a response that is also observed inproteoliposomes containing PL scramblase purified from erythrocytemembranes (J. G. Stout, et al., supra, 1997). A similar acid-dependentactivation of PL mobilizing function was also exhibited byproteoliposomes incorporating recombinant PL scramblase purified from E.coli.

Platelet PL Scramblase. In addition to the presumed role of PLscramblase in PS exposure following cell injury and upon repeatedsickling of SS hemoglobin red cells, the capacity of activated plateletsto rapidly mobilize aminophospholipids across the plasma membrane isthought to play a central role in the initiation of thrombin generationrequired for plasma clotting (R. F. A. Zwaal and A. J. Schroit, Blood89:1121-1132, 1997). Whereas incubation with Ca²⁺ ionophore causes amarked acceleration in transbilayer movement of plasma membrane PL inboth platelets and erythrocytes, the apparent rate of transbilayer PLmigration in platelet exceeds that in erythrocyte by approximately10-fold, implying either a higher abundance of PL scramblase, or theaction of another component in platelet with enhanced PL scramblingfunction (J. C. Sulpice, et al., J. Biol. Chem. 269:6347-6354; 1994; J.C. Sulpice, et al., Biochemistry 35:13345-13352, 1996). Zwaal andassociates recently reported evidence for the existence of protein(s) inplatelet with functional properties similar to that of PL scramblase weisolated from erythrocyte (F. Bassè, et al., supra, 1996; J. G. Stout,et al., supra, 1997; P. Comfurius et al., supra, 1996). In order todetermine whether the protein we now identify in the erythrocytemembrane is also found in platelets, we probed platelets with antibodyagainst PL scramblase residues 306-318. FIG. 4 illustratesimmunoblotting of PL scramblase in human erythrocytes and platelets.2×10⁸ platelets (lane 1), and ghost membranes from 2×10⁸ erythrocytes(lane 2), were separated by SDS-PAGE, transferred to nitrocellulose andWestern blotted with anti-306-318 antibody as described in ExperimentalProcedures. Lane 3 contains 0.9 pmoles of factor Xa cleaved recombinantPL scramblase and lane 4 contains 0.3 pmoles of PL scramblase purifiedfrom erythrocytes. Data of single experiment representative of three soperformed.

As shown in FIG. 4, this antibody blotted a single protein in plateletwith similar mobility to the ˜37 kDa PL scramblase in erythrocyte. Basedon quantitative immunoblotting with anti-306-318, we estimateapproximately 10⁴ molecules/cell in platelet versus 10³ molecules/cellin erythrocyte, consistent with the increased PL scramblase activity andprocoagulant function observed for human platelets versus erythrocytes.

Tissue Distribution. In addition to platelet and red blood cell, PLscramblase activity has been observed in many other cells, and thisCa²⁺-induced response is thought to be central to the rapid movement ofPS and phosphatidylethanolamine from inner plasma membrane leaflet tothe surface of perturbed endothelium, and a variety of injured andapoptotic cells (R. F. A. Zwaal and A. J. Schroit, supra, 1997). Theresulting exposure of PS at the cell surface is thought to play a keyrole in removal of such cells by the reticuloendothelial system, inaddition to activation of both the plasma complement and coagulationsystems (R. H. Wang, et al., J. Clin. Invest. 92:1326-1335, 1993; V. A.Fadok, et al., J. Immunol. 148:2207-2216, 1992; R. F. A. Zwaal and A. J.Schroit, supra, 1997). Whereas the molecular mechanism(s) in eachcircumstance remains unresolved, evidence for a specific plateletmembrane protein functioning to accelerate migration of PL betweenmembrane leaflets at increased cytosolic [Ca²⁺] has been reported (P.Comfurius, et al., supra, 1996), similar to the proposed role of PLscramblase in red blood cells (F. Bassè, et al., supra, 1996; J. G.Stout, et al., supra, 1997). It was thus of interest to determinewhether mRNA for this protein is expressed in nucleated cells where PLscramblase-like activity has been observed.

Northern blotting with PL scramblase cDNA revealed transcripts of ˜1.6and ˜2.6 kb in all tissues and cell lines tested. Some tissue-to-tissueand cell line variability in the relative abundance of these twotranscripts is apparent, the significance of which remains to bedetermined. Also notable was markedly reduced expression in HL-60 andthe lymphoma lines Raji and MOLT-4 whereas abundant message was detectedin spleen, thymus, and peripheral leukocytes. In addition to thetransformed cell lines shown, mRNA for PL scramblase was also confirmedin human umbilical vein endothelial cells. Whereas these data imply thatthe same protein identified as mediating accelerated transbilayerflip-flop of the erythrocyte membrane PL also plays a similar role inthe plasma membrane of platelets, leukocytes and other cells, actualconfirmation for this role of PL scramblase awaits analysis of a cellline that is selectively deficient in this protein. In Scott syndrome, ableeding disorder related to an inherited deficiency of plasma membranePL scramblase function, erythrocytes deficient in PL scramblase activitywere found to contain normal amounts of the PL scramblase protein (J. G.Stout, et al., supra, 1997) and unpublished data). Furthermore, despitethe apparent deficiency in Scott syndrome cells of endogenous PLscramblase function, when PL scramblase protein from these cells waspurified and reconstituted in proteoliposomes containing exogenous PL,it exhibited normal Ca²⁺-dependent PL-mobilizing activity (J. G. Stout,et al., supra, 1997). This suggests that in addition to the knownregulation by intracellular [Ca²⁺], the activity of PL scramblase in theplasma membrane is regulated by other as yet unidentified membrane orcytoplasmic component(s).

EXAMPLE 2 Cloning of Murine PL scramblase and Identity of a ConservedMotif in Phospholipid Scramblase that is Required for AcceleratedTransbilayer Movement of Membrane Phospholipids by Ca²⁺

A. Summary

Accelerated transbilayer movement of plasma membrane phospholipids (PL)upon elevation of Ca²⁺ in the cytosol plays a central role in theinitiation of plasma clotting and in phagocytic clearance of injured orapoptotic cells. We recently identified a human erythrocyte membraneprotein that induces rapid transbilayer movement of PL at elevated Ca²⁺,and presented evidence that this PL scramblase is expressed in a varietyof other cells and tissues where transbilayer movement of plasmamembrane PL is promoted by intracellular Ca²⁺ (Q. Zhou, et al., J. Biol.Chem. 272:18240-18244, 1997). We have now cloned murine PL scramblasefor comparison to the human polypeptide (FIG. 1C and D): Both human andmurine PL scramblase are acidic proteins (pI=4.9) with a predictedinside-outside (type 2) transmembrane segment at the carboxyl-terminus(FIGS. 5A and B). Whereas human PL scramblase (318 AA) terminates in ashort exoplasmic tail, murine PL scramblase (307 AA) terminates in thepredicted membrane-inserted segment. The aligned polypeptide sequencesreveal 65% overall identity, including near identity through 12 residuesof an apparent Ca²⁺ binding motif (D[A/S]DNFGIQFPLD) spanning residues273-284 (human, SEQ ID NO:2) and 271-282 (murine, SEQ ID NO:4),respectively (FIGS. 5A and B). This conserved sequence in thecytoplasmic domain of PL scramblase shows similarity to Ca²⁺-bindingloop motifs previously identified in known EF-hand structures.Recombinant murine and human PL scramblase were each expressed in E.coli and incorporated into proteoliposomes. Measurement of transbilayermovement of NBD-labeled PL confirmed that both proteins catalyzedCa²⁺-dependent PL flip/flop similar to that observed for the action ofCa²⁺ at the cytoplasmic face of plasma membranes. Mutation of residueswithin the putative EF hand loop of human PL scramblase resulted in lossof its PL mobilizing function, suggesting that these residues directlyparticipate in the Ca²⁺ induced active conformation of the polypeptide.

B. Experimental Procedures

Abbreviations used: PL, phospholipids(s); PC, phosphatidylcholine; PS,phosphatidylserine; NBD-PC,1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine;EST, expressed sequence tag; MBP, maltose binding protein; PAGE,polyacrylamide gel electrophoresis; bp, base pair(s); PCR; polymerasechain reaction.

Materials: Mouse fibroblast 5′-stretch plus cDNA library and KlenTaqpolymerase were obtained from CLONTECH Laboratories. Expressed sequenceTag (EST) clone with GenBank™ accession number gb AA110551 was fromAmerican Type Culture Collection (ATCC 977052). α-³²P-dCTP was purchasedfrom Dupont. Random Primed DNA Labeling Kit was from BoehringerMannheim. Hybond-N Nylon membrane was from Amersham. Expression vectorpMAL-C2, amylose resin and all restriction enzymes were from New EnglandBiolabs. Wizard Kit was from Promega. Qiagen Lambda Kit was from Qiagen.Egg yolk phosphatidylcholine (PC), brain phosphatidylserine (PS) and1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine(NBD-PC) were obtained from Avanti Polar Lipids. Factor Xa was fromHaematologic Technologies, and Bio-Beads SM-2 were from BioRad.N-octyl-β-D-glucopyranoside and Glu-Gly-Arg chloromethyl ketone werefrom Calbiochem. Sodium dithionite (Sigma) was freshly dissolved in 1MTris, pH 10, at a concentration of 1 M.

Labeling of DNA Probe: The DNA insert of EST clone gb AA110551 wasreleased by digestion with EcoRI and ApalI and purified by Wizard Kit.Four micrograms of purified DNA were labeled with 1 mCi of α-³²P-dCTP.The specific radioactivity of the probe was 3.9×10⁵ dpm/μg DNA.

Isolation of Mouse PL Scramblase cDNA by Plaque Hybridization: E. colistrain Y1090r was transformed by mouse fibroblast cDNA library (6×10⁵pfu) and poured onto 30 plates (15 cm diameter, 20,000 pfu per plate).Plaques were lifted onto Hybond-N Nylon membranes. After denaturation,neutralization and UV-cross linking, the membranes were firstprehybridized in a solution composed of 5× Denhardt, 5× SSC, 1% SDS, and200 μg/ml herring sperm DNA for 3 hours at 68° C., and then hybridizedin the same solution containing 5 ng/ml ³²P-labeled probe for 16 hoursat 68° C. The membranes were washed once with 2× SSC, 0.1% SDS, thenthree times with 0.1× SSC, 0.1% SDS for 20 minutes at 65° C., andexposed to X-ray film. Secondary plaque lifts and hybridization werecarried out on 8 positive plaques at a density of about 100plaques/plate. Single positive and well isolated plaques were picked andamplified. λDNA was purified with Qiagen Lambda Maxi Kit.

DNA Sequencing. DNA was sequenced on an ABI DNA Sequencer Model 373Stretch (Applied Biosystems) using PRISM Ready Reaction DyeDeoxyTerminator Cycle Sequencing Kit (Perkin Elmer).

Cloning of Mouse PL Scramblase into pMAL-C2 Expression Vector. In orderto express mouse PL scramblase as a fusion protein with maltose bindingprotein (MBP), cDNA encoding mouse PL scramblase was cloned into pMAL-C2expression vector. PCR was performed on a mouse scramblase clone usingthe primers ^(5′)TCA GAA TTC GGA TCC ATG GAG GCT CCT CGC TCA GGA AC^(3′)(SEQ ID NO:8) with an EcoRI site before the ATG start codon and ^(5′)GCTTGC CTG CAG GTC GAC CTA CAC ACA GCC TTC AAA AAA CAT G^(3′) (SEQ ID NO:9)with a SalI site after the stop codon. KlenTaq polymerase was used toensure high fidelity amplification. The PCR product was digested withEcoRI and SalI, isolated by electrophoresis, and cloned into pMAL-C2immediately 3′ of MBP. E. coli strain TB1 was transformed, and sequenceof the cDNA insert of plasmid from a single colony was confirmed.

Expression and Purification of Mouse PL Scramblase-MBP Fusion Protein:Mouse PL scramblase was expressed as fusion protein with MBP in E. coliTB1 and purified on amylose resin as previously described for human PLscramblase (Q. Zhou, et al., J. Biol. Chem. 272:18240-18244, 1997). Thepurified fusion protein was centrifuged at 106,000×g for 1 hour at 4° C.to remove aggregated protein.

Reconstitution and Functional Activity of PL Scramblase: Reconstitution,removal of MBP, and functional assay of PL scramblase were performed aspreviously described (F. Bassè, et al., J. Biol. Chem. 271:17205-17210,1996; Q. Zhou, et al., supra, 1997; J. G. Stout, et al., J. Clin.Invest. 99:2232-2238, 1997). Routinely, 420 pmoles of protein werereconstituted with 1 μmol of PL. To remove MBP, proteoliposomes wereincubated 3 hours at room temperature with {fraction (1/40)} (w/w)factor Xa. The digest was terminated by addition of 100 μM Glu-Gly-Argchloromethyl ketone. Proteoliposomes labeled with NBD-PC were incubatedfor 2 hours at 37° C. in Tris buffer in the presence or absence of CaCl₂as indicated in figure legends and diluted 25-fold in Tris buffercontaining 4 mM EGTA. Initial fluorescence was recorded (SLM Aminco 8000spectrofluorimeter; excitation at 470 nm, emission at 532 nm), 20 mMdithionite was added, and the fluorescence was continuously monitoredfor a total of 120 seconds. Scramblase activity was calculated accordingto the difference in non-quenchable fluorescence observed in presence vsabsence of CaCl₂. Ionized [Ca²⁺] was calculated using FreeCal version4.0 software (generously provided by Dr. Lawrence F. Brass, Universityof Pennsylvania, Philadelphia, Pa.).

Protein Concentrations: Protein concentrations were estimated based uponoptical density at 280 nm, using extinction coefficients (M⁻¹cm⁻¹) of39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scramblase-MBPfusion protein).

Mutagenesis of PL Scramblase: Human PL scramblase amino acid residues inEF-hand Ca²⁺-binding motif at positions of Asp²⁷³, Asp²⁷⁵, Phe²⁷⁷,Ile²⁷⁹, Phe²⁸l and Asp²⁸⁴ were mutated to Ala witholigonucleotide-directed mutagenesis by two rounds of PCR. PLscramblase-pMAL-C2 was selected as template, and the first round of PCRwas performed with pairs of a complementary oligonucleotide primercontaining the point mutation plus a primer complementary to a site nearthe ATG initial codon or TAG stop codon. PCR products were purified byWizard kit. Full length mutated PL scramblase cDNA was obtained byoverlapping PCR and cloned back into pMAL-C2 vector. After confirmationof correct DNA sequence the mutants were recombinantly expressed in E.coli as described above and analyzed by SDS-PAGE.

C. Results and Discussion

Isolation of cDNA of Mouse PL Scramblase. Murine EST clones in GenBankcontaining putative PL scramblase sequence were identified by a Blasthomology search using the human PL scramblase cDNA. Among several clonesexhibiting significant homology, a 403 bp Stratagene mouse kidney clone(gb accession number AA110551) with 79% nucleotide sequence identity tohuman PL scramblase was selected and this clone was used to probe amouse fibroblast cDNA library. Eight positive clones were identifiedafter two rounds of plaque hybridization. Two of the eight clones weresequenced yielding 1354 bp and 1529 bp, respectively. Alignment revealed1261 bp of overlapping sequence that spanned an open reading frame of921 bp and specified a total of 1622 bp of unique cDNA sequence (SEQ IDNO:3).

SEQ ID NO:4 represents the open reading frame of the translated sequenceof SEQ ID NO:3 (see FIGS. 1C and D). The deduced mouse PL scramblasecDNA encodes a 307 residue protein with a molecular weight of 33.9 kDaand a theoretical pI=4.9, similar to values obtained for the humanprotein (318 residues, 35.1 kDa; pI=4.9; ref. (Q. Zhou, et al., supra,1997). The overall identity of the mouse and human PL scramblase is64.8%, with the most divergent sequence generally contained in theN-terminal portion of the polypeptide (FIGS. 5A and B). FIG. 5 depictsthe alignment of protein sequences of mouse and human PL scramblase.Alignment between mouse (MUR) and human (HUM) PL scramblase wasperformed by FASTA program using the Smith-Waterman algorithm. (W. R.Pearson and D. J. Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988)Sequence of human PL scramblase is contained in GenBank T accessionnumber AF008445. Amino acid identities (:) or similarities (.) betweenthe two sequences are indicated. Also indicated are the residuescomprising a predicted inside-out transmembrane domain (MUR 289-307, HUM291-309; double underline), and the 12 residues of the acidic loop of aputative EF-hand (MUR 271-282, HUM 273-284; single underline).

In both proteins, a single 19 residue transmembrane helix is predictedat the carboxyl terminus, exhibiting a strongly preferredinside-to-outside orientation. Whereas the mouse protein terminatesimmediately after this conserved transmembrane helix, the human PLscramblase contains an additional nine residues, implying that the shortexoplasmic peptide in human PL scramblase is non-essential to function.Homology motifs conserved in both proteins include a potential site forprotein kinase C phosphorylation (Thr¹⁵⁹ in mouse, Thr¹⁶¹ in human) anda potential Ca²⁺-binding EF-hand loop motif adjacent to thetransmembrane helix (residue Asp²⁷¹ to Asp²⁸² in mouse and residuesAsp²⁷³ to Asp²⁸⁴ in human). The cytoplasmic orientation of this proteinand the proximity of this putative Ca²⁺-binding domain to the segment ofpolypeptide that is inserted into the plasma membrane are consistentwith the proposed activity of this protein in situ, where Ca²⁺ actingdirectly at the endofacial membrane surface is known to initiate therapid transbilayer movement of plasma membrane PL (P. Williamson, etal., Biochemistry 31:6355-6360, 1992; R. F. A. Zwaal, and A. J. Schroit,Blood 89:1121-1132, 1997; F. Bassè, et al., J. Biol. Chem.271:17205-17210, 1996; D. L. Bratton, J. Biol. Chem. 269:22517-22523,1994; B. Verhoven, et al., Biochim. Biophys. Acta 1104:15-23, 1992).

Functional Activity of Recombinant Mouse PL Scramblase. In order toconfirm that the cDNA identified as mouse PL scramblase encodes aprotein of similar function to that identified in human, the human andmouse proteins were each expressed in E. coli, purified, andreconstituted in proteoliposomes for measurement of PL mobilizingactivity. Mouse or human PL scramblase-MBP fusion protein (420 pmoles)was reconstituted into PC/PS liposomes (1 μmol total PL), respectively,MBP was removed by digestion of the proteoliposomes with factor Xa, andPL scramblase activity was determined as described under “ExperimentalProcedures” and plotted as a function of external free [Ca²⁺]. Theresults of this experiment indicate that recombinant mouse PL scramblasemediated a Ca²⁺-dependent transbilayer movement of membrane PL with aspecific activity and affinity for Ca²⁺ indistinguishable from thatobserved for the recombinant human protein.

Mutational Analysis of a Putative Conserved EF-Hand Motif. As notedabove, the deduced protein sequence of mouse and human PL scramblasereveals an extensive segment of highly conserved sequence extendingthrough residue Glu³⁰⁶ (in human; corresponding to Glu³⁰⁴ in mouse; FIG.5). The predicted secondary structure through this portion of theprotein reveals that it contains two short alpha-helical segments nearthe C-terminus that are separated by a 12-residue acidic loop. In bothproteins (human and mouse), the C-terminal alpha helix represents apredicted transmembrane segment with a strongly-preferredinside-to-outside orientation, whereas sequence contained within theadjacent 12-residue acidic loop conforms in-part to a consensus sequencethat is characteristic of an EF-hand Ca²⁺-binding loop motif (S.Nakayama and R. H. Kretsinger, Annu. Rev. Biophys. Biomol. Struct.23:473-507, 1994). In this motif, residues in positions 1, 3, 5, 7, 9and 12 of the loop contribute to octahedral coordination of the Ca²⁺ion, with the residues in position 1 [Asp], 3 [Asp, Asn, or Ser] and 12[Asp or Glu] being those most highly conserved.

In order to gain insight into whether this segment of the protein mightbe directly involved in the Ca²⁺-dependent reorganization of membrane PLmediated by PL scramblase, we expressed mutant human PL scramblase withAla substitutions at positions corresponding to residues 1 (Asp²⁷³), 3(Asp²⁷⁵ ), 5 (Phe²⁷⁷), 7 (Ile²⁷⁹), 9 (Phe ²⁸¹) and 12 (Asp²⁸⁴) of thisputative 12 residue EF-hand loop. FIG. 6 illustrates PL scramblaseactivity as a function of mutational analysis of putative EF hand loopmotif contained in human PL scramblase. Wild-type (WT) and mutantconstructs of human PL scramblase were expressed as fusion proteins withMBP in E. coli, purified, and reconstituted in proteoliposomes. Afterrelease of MBP by incubation with factor Xa, PL scramblase activity wasassessed (see “Experimental Procedures”). For each mutant construct, theresidues in human PL scramblase that were replaced by Ala are indicatedon the abscissa. PL scramblase activity (ordinate) was measured inpresence of 2 mM CaCl₂, and in each case was normalized to the activityof WT human PL scramblase (11.76±0.44% of total NBD-PC flipped), withcorrection for the non-specific transbilayer movement of NBD-PC(0.20±0.08% of total NBD-PC flipped) measured in PL vesicles lackingadded protein. Error bars indicate mean ± SD of three independentmeasurements performed with each mutant construct. FIG. 6 illustratesthe data of single experiment, representative of two separateexperiments so performed.

As illustrated by FIG. 6, Ala substitution at any of these positionsreduced PL scramblase function, with mutation at Asp²⁷⁵ resulting incomplete inactivation of the Ca²⁺-dependent response. In those mutantpolypeptides showing partial retention of activity, reduced response toCa²⁺ was related in-part to an apparent reduction in avidity for Ca²⁺(FIG. 7). FIG. 7 illustrates the Ca²⁺-dependence of mutant human PLscramblase. PL scramblase activity of wild-type (WT) and selected mutantconstructs of FIG. 6 was determined as described in “ExperimentalProcedures” and plotted as a function of external free [Ca 2+] WT();Asp²⁷³(□); Phe²⁷⁷ (Δ); Ile²⁷⁹(⋄); Phe²⁸¹ (∘) Asp²⁸⁴(∇). The data arecorrected for non-specific transbilayer migration of NBD-PC in theabsence of free [Ca²⁺]. Data of single experiment. The results describedin FIG. 7 suggest that residues contained in the putative EF-hand loopspanning Asp²⁷³-Asp²⁸⁴ are critical to the function of PL scramblase,presumably for coordination of Ca²⁺ as required to induce the PLtransporting state of the protein. It remains to be determined whatconformational changes are induced in the polypeptide in the presence ofCa²⁺, including potential reorientation of helical segments flanking theputative Ca²⁺ binding loop, that might contribute to the acceleratedtransbilayer movement of membrane phospholipids.

EXAMPLE 3 Plasma Membrane Expression of Phospholipid ScramblaseRegulates Ca²⁺ Induced Movement of Phosphatidylserine to the CellSurface: Alteration of Phosphatidylserine Exposure In Human LymphoblastsThrough Stable Transfection with PL Scramblase cDNA

A. Summary

In order to determine whether PL scramblase is responsible for the rapidmovement of PS from inner-to-outer plasma membrane leaflets in othercells exposed to elevated cytosolic [Ca²⁺]_(c), we analyzed how inducedmovement of PS to the surface related to cellular content of PLscramblase. Exposure to Ca²⁺ ionophore A23187 resulted in rapid PSexposure in those cells high in PL scramblase (K-562, HEL, 293T, andEBV-transformed lymphocytes), whereas this response was markedlyattenuated in cells with low amounts of the protein (Raji, MOLT-4,HL-60). To confirm this apparent correlation between PL scramblaseexpression and PS egress at elevated [Ca²⁺]_(c), Raji cells weretransfected with PL scramblase cDNA in pEGFP-C2, and stabletransformants expressing various amounts of rGFP-PL scramblase fusionprotein obtained. Clones expressing rGFP-PL scramblase showed plasmamembrane-localized fluorescence and elevated PL scramblase antigenwhereas clones expressing rGFP alone (transfected with pEGFP-C2 withoutinsert) showed only cytoplasmic fluorescence and served as controls. Inabsence of ionophore, expression of rGFP-PL scramblase had no effect oncell viability or background PS exposure. In response to A23187, clonesexpressing GFP-PL scramblase exhibited markedly accelerated movement ofPS to the cell surface when compared to A23187-treated clones expressingGFP with PS movement to the cell surface increasing with amount ofrGFP-PL scramblase expressed. These data indicate that transfection withPL scramblase cDNA promotes [Ca²⁺]_(c)C-dependent movement of PS to thecell surface and suggest that this protein normally mediatesredistribution of plasma membrane phospholipids in activated, injured,or apoptotic cells exposed to elevated [Ca²⁺]C.

B. Materials and Methods

Materials. All restriction enzymes were from New England BioLabs, Inc.(Beverly, Mass.). Klentaq polymerase and pEGFP-C2 vector were fromCLONTECH Laboratories (Palo Alto, Calif.). Bovine coagulation factor Va(FVa), factor Xa (FXa), prothrombin anddansylarginine-N-(3-ethyl-1,5-pentanediyl)amide were from HaematologicTechnologies, Inc. (Essex Junction, Vt.). Chromogenic thrombin substrateS2238 was from DiaPharma Group, Inc. (Franklin, Ohio). Human a-thrombinwas a generous gift from Dr. John W. Fenton (Albany, N.Y.). OPTI-MEM andgeneticin were from Life Technologies (Gaithersburg, Md.). Fetal bovineserum, RPMI 1640, Cell Dissociation Solution, Hank's Balanced SaltSolution (HBSS), Protein A Sepharose-CL4B, leupeptin, and BSA were fromSigma Chemical Co. (St. Louis, Mo.). UltraLink Iodoacetyl resin andSuperSignal ULTRA Chemiluminescence Kit were from Pierce Chemical Co.(Rockford, Ill.). All other chemicals were of reagent grade.

Cell culture: Human cancer cell lines erythroleukemic HEL, promyelocyticleukemia HL-60, chronic myelogenous leukemia K562, lymphoblasticleukemia MOLT-4, acute T-cell leukemia Jurkat, Burkitt's lymphoma Raji,and megakaryocytic DAMI were from American Type Culture Collection(Rockville, Md.) and cultured in RPMI 1640 containing 10% fetal bovineserum. EBV-transformed cell line W9 established from peripheralB-lymphocytes of a normal donor was maintained as previously described(H. Kojima, et al., 1994).

Antibodies: Anti-GFP: murine monoclonal antibody against greenfluorescent protein (GFP) was from CLONTECH Laboratories. Anti-FVa:murine monoclonal antibody V237 reactive against human or bovine factorVa light chain was the generous gift of Dr. Charles T. Esmon (OklahomaMedical Research Fndn, Oklahoma City, Okla.). Anti-PLScramblase-E306-W318: Rabbit antibody raised against the carboxylterminal peptide sequence E306-W318 of human PL scramblase haspreviously been described (Q. Zhou, et al., supra, 1997). The IgGfraction was isolated on protein A-Sepharose-CL4B and thepeptide-reactive antibody purified by affinity chromatography on peptide[Cys]-ESTGSQEQKSGVW (SEQ ID NO:5) coupled to UltraLink Iodoacetyl resin.

Plasmid Construction: Human PL scramblase cDNA insert was released fromplasmid pMAL-C2-PL scramblase (Q. Zhou, et al., supra, 1997) by doublecutting with EcoRI and SalI, respectively, and then ligated intopEGFP-C2 vector using the same restriction site. The pEGFP-C2-PLscramblase plasmid was amplified from single clones in E. coli strainTop 10, and the orientation and reading frame of the insert confirmed bysequencing on an ABI DNA Sequencer Model 373 Stretch (PerkinElmer-Applied Biosystems, Foster City, Calif.) using PRISM ReadyReaction DyeDeoxy Terminator Cycle Sequencing Kit.

Transfection of Raji cells with pEGFP-PL scramblase. 1.6×10⁷ Raji cellswere electroporated with 160 μg plasmid DNA (pEGFP-C2-PL scramblase orpEGFP-C2) in a total volume of 0.8 ml OPTI-MEM, using Gene PulseElectroporator (Bio-Rad Laboratories, Hercules, CA) set at 450 V, 500μF. After 48 hours in culture, 1.5 mg/ml geneticin was added to themedium and continuously maintained for 4 weeks. Stable transformantsexhibiting GFP fluorescence were sorted by flow cytometry (FACStar,Becton-Dickinson Immunocytometry Systems, San Jose, Calif.) using an FL1sorting gate. The FL1-positive cells were dilutionally cloned in 96 wellculture plates. PEGFP-PL scramblase transformants expressing the 62 kDaGFP-PL scramblase fusion protein were identified by Western blottingwith anti-GFP and with anti-PL-306-W318 antibodies. Western blotting ofpEGFP-C2 transformants (without insert) confirmed presence of 27 kDaGFP. Clones expressing various amounts of GFP-PL scramblase were eachexpanded for functional assay, along with comparable GFP-expressingclones serving as controls.

Fluorescence Microscopy. Cell clones transfected with pEGPF-C2-PLscramblase or pEGFP-C2 were deposited on glass microscopy slides using aCytospin 3 (Shandon, Inc., Pittsburgh, Pa.). Phase contrast andfluorescence microscopy was performed with a ZEISS AXIOSKOP microscope(Carl Zeiss, Inc., Thornwood, N.Y.) equipped for epifluorescence, andimages were recorded with a MC100 camera system. The exposure times forphotography of fluorescence was 80-200 seconds under automatic controlusing Kodak Ektachrome 1600 film.

Western Blot Analysis. Western blotting of GFP and PL scramblaseantigens was performed using 1.5×10⁶ cells per lane. After washing inHBSS, supernatants were removed, and the cell pellets extracted with 2%(v/v) NP-40 in 5 mM EDTA, 50 mM benzamidine, 50 mM N-ethyl maleimide, 1mM phenylmethylsulfonyl fluoride, 1 mM leupeptin in HBSS. After removalof insoluble material (250,000×g, 30 minutes, 4° C.), the samples weredenatured at 100° C. in 10% (w/v) SDS sample buffer containing 2%β-mercaptoethanol. Following SDS-PAGE and transfer to nitrocellulose,the blocked membrane was incubated with either 10 μg/ml of rabbitanti-PL scramblase-E306-W318, or {fraction (1/10,000)} dilution of mouseanti-GFP. The blots were developed with the horseradish peroxidaseconjugate of either goat anti-rabbit IgG or goat anti-mouse IgG,respectively, using SuperSignal ULTRA chemiluminescence.

Measurement of cell surface PS. Calcium ionophore-induced exposure of PSon the surface of all cell lines analyzed was detected by the specificbinding of coagulation factor Va (light chain) as previously described(P. J. Sims, et al., J. Biol. Chem. 263:18205-18212, 1988; H. Kojima, etal., J. Clin. Invest. 94:2237-2243, 1994). Briefly, cells were washedtwice to remove serum proteins and suspended (2×10⁶ cells/ml) at 37° C.in RPMI 1640 supplemented with 0.1% BSA, 20 mM HEPES, and adjusted to1.2 mM free [Ca²⁺]. At time=0, A23187 (0 or 2 μM final concentration)was added from 1 mM stock solution in DMSO, and at times indicated infigure legends, the reaction was stopped by addition of 6 mM EGTA. PSexposed on the cell surface at each time point was detected byincubating (10 minutes, room temperature) 50 μl of the cell suspensionwith 10 μg/ml FVa, followed by 10 μg/ml anti-FVa, to detect thecell-bound FVa light chain. After staining with 10 μg/ml Tri-Colorconjugated goat anti-mouse IgG (CALTAG Laboratories, Burlingame,Calif.), single-cell fluorescence was quantitated by flow cytometry (FL3channel, FACScan, Becton Dickinson Immunocytometry Systems). Use ofTri-Color conjugate to detect cell-bound FVa enabled simultaneousmeasurement of cell-associated GFP fluorescence in cell linestransformed with the pEGFP-C2 expression plasmid (fluorescence of GFPdetected in FL1 channel). In experiments in which cell lysis wasmonitored by uptake of propidium iodide, cells were stained for boundFVa with FITC-conjugate of goat anti-mouse IgG (FL1 channel)substituting for Tri-Color conjugate, and propidium iodide was detectedin FL3 channel. Propidium iodide (0.5 μg/ml) was added immediatelybefore dilution for flow cytometry.

Prothrombinase Assay. Prothrombinase activity of Raji cells wasdetermined by modification of methods previously described forplatelets, using the chromogenic thrombin substrate S2238 (P. J. Sims,et al., supra, 1988). Briefly, 1×10⁵ Raji cells (transfected with eitherpEGFP-C2 or pEGFP-C2-PL scramblase) were suspended in 200 μl HBSScontaining 1% BSA in the presence of 2 nM FVa, 1.4 μM prothrombin 2.5 mMCaCl₂, and 4 μM dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (toinhibit feed-back activation by thrombin), and incubated at 37° C.Ca²⁺-ionophore A23187 (2 μM), or DMSO (as solvent control) was added,and prothrombin conversion was initiated by addition of 2 nM Fxa.Thrombin generation was stopped after 2 minutes by dilution into 10 mMEGTA and samples were stored on ice. Aliquots were transferred to a96-well plate, and thrombin generated was assayed in TBS containing 1%BSA in presence of 150 PIM S2238 by monitoring time-dependent changes inabsorbance at 405 nm using a Thermomax plate reader (Molecular Devices,Sunnyvale, Calif.). Thrombin activity was calculated using purifiedthrombin as standard.

C. Results

Analysis of PL scramblase in various human cell lines. Proteoliposomesreconstituted with erythrocyte PL scramblase exhibit acceleratedtransbilayer movement of fluorescent phospholipids in response to addedCa²⁺, similar to the observed effect of calcium on the endofacialsurface of the red cell membrane (Q. Zhou, et al., supra, 1997; J. G.Stout, et al., J. Clin. Invest. 99:2232-2238, 1997; Bassè, et al., J.Biol. Chem. 271:17205-17210, 1996). In order to determine whether thissame protein is responsible for mediating the accelerated egress ofplasma membrane PS that is observed under conditions of elevatedcytosolic [Ca²⁺]_(c), we undertook to determine whether the level ofexpression of PL scramblase in various human cell lines correlated tothe induced movement of PS to the surface of these cells. Whenchallenged with a calcium ionophore, human cell lines exhibitconsiderable differences in the extent to which PS is mobilized to thecell surface. Among the cells tested, Raji, HL60, and Dami were notablyunresponsive to A23187, whereas HEL, W9 (an EBV-transformed normalB-lymphocyte), and Jurkat showed notably robust responses. This apparentcell type-specific variability in response to induced elevation of[Ca²⁺]_(c) was consistently maintained through many months of passage inculture, suggesting it reflected an inherent property of each cell line.As shown in FIG. 8, we also observed considerable differences in thecontent of PL scramblase protein among these various cell lines, and thesensitivity of these various cell lines to induced exposure of plasmamembrane PS (lower panel) generally correlated with the amount ofcellular PL scramblase protein detected by Western blotting (upperpanel): Those cell lines that were most responsive to induced elevationof [Ca²⁺]_(c) (HEL, W9, Jurkat) also expressed greatest amounts of PLscramblase antigen, whereas cell lines with attenuated response to[Ca²⁺]_(c) (Raji, HL60, Dami) contained relatively little of thisprotein. Cell lines Molt-4 and K562 showed intermediate responses toelevated [Ca²⁺]_(c) and expressed intermediate levels of PL scramblaseantigen.

FIG. 8 depicts western blot analysis of PL scramblase in various humancell lines. Constitutive expression of PL scramblase was analyzed in thehuman cell lines indicated. Upper Panel: Results obtained by Westernblotting with antibody specific for PL scramblase carboxyl terminalresidues E306-W318 (see Materials & Methods). Each lane contains thetotal protein extract of 1.5×10⁶ cells. Lower Panel: Cumulative resultsof three separate experiments performed as follows: The cells indicatedwere washed and suspended at 37° C. in the presence of 1.2 mM free Ca²⁺,and 2 μM A23187 was added. At times shown (abscissa), EGTA was added andcells analyzed for surface exposed PS as detected by cell-bound FValight chain (see Materials and Methods). Data plotted represent the meanincrease (± SD) in number of cells that stained positive for surface PSafter 5 minutes incubation with ionophore, after correction for initialbackground of PS-positive cells before addition of ionophore (time=0).Background number of cells that exposed PS in absence of ionophore wasalways <15% except in case of HEL, where this background ranged between15-30%.

These relatively large differences in cell line-specific expression ofthis protein was also consistently observed despite repeated passage inculture, and was found to correspond to marked differences in level ofspecific mRNA as detected by Northern blotting with PL scramblase cDNA(Q. Zhou, et al., supra, 1997), and data not shown). We also noted thatthose cell lines with the highest content of PL scramblase generallyexhibited a higher background of PS exposed on the surface in absence ofadded ionophore. This was most notable for HEL for which approximately15-30% of the cells were consistently found to expose PS prior toaddition of A23187 (see Discussion).

Membrane chances underlying ionophore response. In order to determinewhether the increase in PS exposure in ionophore-treated cells reflectedfacilitated movement of PS from inner to outer leaflets of the plasmamembrane, or, greater sensitivity of the plasma membrane to lyticdisruption, FVa binding to the cell surface was monitored simultaneouslywith uptake of propidium iodide, as a measure of cell lysis. Asillustrated for the human B-lymphocyte lines W9 (high content of PLscramblase) and Raji (low content of PL scramblase), the inducedmovement of PS to the cell surface was found to precede uptake ofpropidium iodide, suggesting that the elevation of [Ca²⁺], induces acollapse of transmembrane PL asymmetry before onset of lysis. In thecase of Raji cells which are virtually devoid of PL scramblase (see FIG.8), a general insensitivity of the plasma membrane to eitherionophore-induced PS exposure or to lysis was also apparent.

Transfection of the Raji cell line with pEGFP-C2-PL scramblase. In orderto confirm that the extent to which PS moves to the cell surface withelevation of [Ca²⁺], actually depends upon the plasma membrane contentof PL scramblase, we stably transformed Raji, a cell line exhibiting lowendogenous PL scramblase expression by transfection with plasmidpEGFP-C2-PL scramblase. This plasmid expresses PL scramblase as a fusionprotein with green fluorescent protein (GFP), facilitating flowcytometric sorting of transformants for subsequent cloning and detectionof the expressed recombinant protein in selected clones. The decision toattach GFP to the amino terminus of PL scramblase was based on priorevidence that the carboxyl terminus of the protein is membrane insertedand essential for function, and the observation that other aminoterminal fusion constructs of PL scramblase expressed in E. coliretained the same activity of the unmodified PL scramblase polypeptidewhen reconstituted in proteoliposomes (Q. Zhou, et al., supra, 1997),and unpublished data). The expression of the full-length GFP-PLscramblase fusion protein in selected transformed clones was confirmedby Western blotting with antibody specific for GFP, and with antibodyraised against peptide sequence of the carboxyl terminus of human PLscramblase. As illustrated by fluorescence micrographs shown in FIG. 9,clones that expressed the GFP-PL scramblase fusion protein showed adistinct rim appearing pattern of fluorescence, consistent withtrafficking of GFP-PL scramblase to the plasma membrane. FIG. 9illustrates fluorescence micrographs of GFP-PL scramblase transformedRaji cells. Fluorescence photomicrography of GFP fluorescence expressedin the transformed Raji clones was performed as described in Materialsand Methods. FIG. 9A shows fluorescence of cells expressing GFP; FIG. 9Bshows cells transfected with pEGFP-C2-PL scramblase plasmid andexpressing GFP-PL scramblase fusion protein. Data of single experiment,representative of results obtained for all clones transfected witheither pEGFP-C2 or pEGFP-C2-PL scramblase. By contrast, clones thatexpressed GFP alone exhibited diffuse fluorescence throughout thecytoplasm, with no obvious staining of the plasma membrane. These dataprovide the first direct evidence that PL scramblase cDNA encodes aprotein that predominantly trafficks to the plasma membrane under normalconditions of cell growth.

Analysis of PS mobilizing function in GFP-PL scramblase transformants.After geneticin selection, clonal populations of transformed Raji cellsexpressing comparable levels of either GFP-PL scramblase or GFP(transformed with pEGFP-C2 lacking insert) were analyzed for theircapacity to mobilize PS to the cell surface. In response to anA23187-induced elevation of [Ca²⁺]_(c), transformants expressing theGFP-PL scramblase fusion construct showed a marked increase in both therate and extent that PS became exposed on the cell surface, whencompared to either the identically-treated parental Raji cell line or toGFP-expressing clones transformed with pEGFP-C2 vector alone. As wasalso evident from these data, in the absence of ionophore, weconsistently noted a small but reproducible increase in the backgroundlevel of PS exposure in transformants expressing GFP-PL scramblaseprotein, when compared to either the parental Raji cell lines or toGFP-expressing clones transformed with vector alone.

Induction of membrane procoagulant function through expression of PLscramblase. In order to confirm that the increased expression of FVabinding sites detected upon activation of GFP-PL scramblase transformedclones reflected an increase in the procoagulant (clot-promoting)properties of the plasma membrane of these cells, the capacity of GFP-PLscramblase transformed cells to provide catalytic membrane surface forthe prothrombinase (FVaXa) enzyme complex was compared to clonesexpressing GFP alone. These data confirmed that expression ofrecombinant PL scramblase in the Raji cell line was also accompanied byan increase in cell capacity to catalyze the prothrombinase reactionupon entry of calcium into the cytosol.

Level of expression of PL scramblase regulates capacity of to mobilizePS to the cell surface. In order to confirm the apparent correlationbetween endogenous cell content of PL scramblase and plasma membranesensitivity to elevated [Ca²⁺]_(c) that is evident when different humancell lines are compared (see FIG. 8), we analyzed multiple Raji clonesthat were stably transfected with either GFP-PL scramblase or with GFPvector alone (FIG. 10). FIG. 10 illustrates that the level of expressionof PL scramblase determines plasma membrane sensitivity to intracellularCa²⁺. The relationship between level of recombinant protein expressed(GFP fluorescence detected in FL1 channel; abscissa) and numbers ofcells that expose PS after 2 minutes incubation with A23187 (ordinate)is plotted for multiple transformed Raji clones. Analysis was gated toinclude only those cells distinctly positive for GFP fluorescence (FL1channel), and cell-bound FVa was stained with Tri-color conjugate anddetected in FL3 channel (see Materials and Methods). Analysis wasperformed on all cells positive for GFP fluorescence. ; Open symbolsindicate individual clones stably transformed by transfection withpEGFP-C2; closed symbols indicate individual clones stably transformedwith pEGFP-PL scramblase. Data of single experiment, representative ofthree so performed. These experiments confirm that the capacity ofGFP-PL scramblase transformants to mobilize PS to the cell surfacegenerally correlates with the amount of the expressed GFP-PL scramblasefusion protein, whereas this cell response to increased [Ca²⁺]_(c) isunaffected by cell content of GFP. In addition to confirming the role ofPL scramblase in the plasma membrane response to [Ca²⁺]_(c), these datasuggest that the capacity to mobilize PS to the cell surface and therebysupport plasma clotting in activated, injured or apoptotic cells exposedto elevated [Ca²⁺]_(c) can be altered by changing the level ofexpression of PL scramblase expressed in the plasma membrane.

Discussion

These results provide the first evidence that the PL scramblase proteinidentified in the erythrocyte membrane and implicated in[Ca²⁺]_(c)-induced remodeling of membrane phospholipids actuallyfunctions to induce accelerated transbilayer movement of plasma membranephospholipid in human cells that express this protein. Our results alsoconfirm that the level of expression of plasma membrane PL scramblasecan determine the extent to which PS is mobilized to the cell surfaceupon elevation of [Ca²⁺]_(c), and suggest that this protein normallyfunctions to mediate the redistribution of plasma membrane phospholipidsin response to the entry of calcium into the cytosol. Furthermore, thesedata provide the first indication that the movement of PS and otherprocoagulant aminophospolipids from plasma membrane inner leaflet to thecell surface can be manipulated by selectively altering the level ofexpression of a particular cellular protein, either through directtransfection with the PL scramblase cDNA, or potentially, by anotherintervention affecting cellular expression of functional PL scramblaseprotein.

Whereas these experiments suggest that direct activation of plasmamembrane PL scramblase is responsible for the increased cell surfaceexposure of PS that is observed in various activated, injured orapoptotic cells exposed to elevated [Ca²⁺]_(c), we cannot exclude thepossibility that there are other cellular components that contribute tothe accelerated movement of PS from inner to outer plasma membraneleaflet under these conditions. In particular, whereas PL scramblase hasbeen shown to mediate the bidirectional movement of PS and otherphospholipids between membrane leaflets, it has been suggested thatthere is also a PS-selective pathway in the platelet plasma membrane,designated “PS floppase”, which mediates vectorial movement of PS frominner to outer plasma membrane leaflet (P. Gaffet, et al., Biochemistry34:6762-6769. 1995). Experimental evidence for the existence of thisvectorial and headgroup-selective PS floppase pathway in platelet orother cell membranes remains controversial (R. F. A. Zwaal, et al.,supra, 1997; P. Williamson, et al., Biochemistry 31:6355-6360, 1995; C.-P. Chang, et al., J. Biol. Chem. 268:7171-7178, 1993), and awaitsidentity of a [Ca²⁺]_(c)-activated and PS-selective transporter that isdistinct from the plasma membrane PL scramblase found in platelets anderythrocytes, a protein that does not exhibit apparent selectivity forthe PS headgroup (J. G. Stout, et al., supra, 1997; P. Comfurius, etal., Biochemistry 35,7631-7634, 1996; F. Bassè, et al., supra, 1996).

In addition to conferring increased sensitivity of the plasma membraneto ionophore-induced elevation of [Ca²⁺]_(c), we generally observed ahigher background of PS exposure (in absence of ionophore) in thosetransfected cell clones expressing large amounts of the GFP-PLscramblase fusion protein. This elevated background PS exposure was alsoobserved in the case of untreated HEL, the cell line containing thehighest endogenous content of PL scramblase. Although we suspect thatthis increased background reflects the enhanced sensitivity of theplasma membrane of these cells to any adventitial elevation of[Ca²⁺]_(c) during cell processing for assay, we cannot exclude thepossibility that these cells are also inherently more fragile due to thelarge amounts of PL scramblase that is inserted into the plasmamembrane.

While the movement of plasma membrane PS to the cell surface at elevated[Ca²⁺]_(c) can be demonstrated in a variety of cells and tissues (R. F.A. Zwaal, et al., supra, 1997; P. Devaux, supra, 1991), we detect markeddifferences in the levels of PL scramblase mRNA and protein amongdifferent human cell types, which is generally reflected bycorresponding differences in sensitivity to this [Ca²⁺]-induced collapseof plasma membrane PL asymmetry (see FIG. 8, and Q. Zhou, et al., supra,1997). Although the transcriptional regulation of the PL scramblase generemains to be determined, it is of interest to note that such cell ortissue-specific differences in PL scramblase expression has thepotential to significantly affect the biological properties of the cell.In particular, we note that the content of PL scramblase in humanplatelet is approximately 10-fold greater than that of the erythrocyte,which is consistent with the respective PS-mobilizing potential anddifferent roles of these two cells in contributing procoagulant membranesurface for thrombin generation during blood clotting (Q. Zhou, et al.,supra, 1997). In addition to the relatively high levels of PL scramblaseidentified in circulating human platelets, this protein was also mostabundant in the cell line HEL, whereas only small amounts of thisprotein (and low PL scramblase activity) was detected for Dami (FIGS.8), two human cancer cell lines exhibiting partial megakaryocytic-likeproperties. It is also noteworthy that several of the lymphoma-derivedcell lines (e.g., Raji, MOLT-4) express considerably reduced levels ofPL scramblase, and also show distinctly attenuated PS exposure inresponse to elevated [Ca²⁺]_(c), when compared to either peripheralblood leukocytes or to EBV-transforms of normal lymphocytes (FIG. 8).The collapse of plasma membrane phospholipid asymmetry is a relativelyearly event in apoptosis of lymphocytes and other cells, and theconsequent exposure of PS on the cell surface is thought to contributeto phagocytic removal of such cells by scavenger macrophages (V. A.Fadok, et al., J. Immunol. 148:2207-2216, 1992; B. Verhoven, et al., J.Exp. Med. 182:1597-1601, 1995). It is therefore of interest to considerwhether the apparent resistance of certain lymphoma-derived cell linesto such [Ca²⁺]_(c)-induced remodeling of plasma membrane phospholipidsmight contribute to the proliferative potential and in vivo survival ofthese or other transformed cells.

EXAMPLE 4 Inactivation of Human PL Scramblase by Treatment With theThiolester Cleaving Reagent, Hydroxylamine

A. Summary

Incubation of human erythrocyte PL scramblase with hydroxylamine underconditions known to favor hydrolysis of protein cysteinyl-fatty acylbonds was found to cause near complete loss of PL scramblase's functionin promoting movement of PL between membrane leaflets. These datasuggest that for normal activity, the PL scramblase polypeptide requirespost translational modification through addition of a thiolester-linkedfatty acid. Furthermore, these data imply that methods that eitherprevent cellular acylation of the polypeptide, or that cleave cysteinylthiolester linkages, will effectively inhibit endogenous PL scramblaseactivity.

B. Methods

Protein purification. PL scramblase was purified from human erythrocyteghost membranes as previously described (F. Bassè, et al.,supra, 1996;J. G. Stout, et al., supra, 1997).

Treatment with Hydroxylamine. PL scramblase was incubated 1 hour roomtemperature in 1 M hydroxylamine, 25 mM octylglucoside, 1 M Tris-HCl atpH 7.4. Match control samples of the protein were identically incubatedunder these conditions, omitting hydroxylamine. After incubation,samples were dialyzed and reconstituted into PL proteoliposomes forassay of PL scramblase activity.

Membrane reconstitution and assay. PL scramblase was reconstituted intoproteliposomes and activity determined as previously described(F. Bassè,et al., supra, 1996; J. G. Stout, et al, supra, 1997).

C. Results and Discussion

As shown in FIG. 11, incubation with hydroxylamine resulted in nearlycomplete inactivation of PL scramblase. FIG. 11 illustrates inactivationof PL scramblase by hydroxylamine. Purified human erythrocyte PLscramblase was incubated 1 hour, room temperature, in the presence of 50mM octylglucoside, 1M TrisHCl, and either 0 (control) or 1M(hydroxylamine) hydroxylamine at pH 7.2. Untreated refers to samplemaintained in low ionic strength sample buffer at 4° C. Each sample wasthen dialyzed and reconstituted into proteoliposomes for assay of PLscramblase activity using NBD-PC as detailed in Bassè, et al., supra,1996 with modifications of Stout, et al., supra, 1997. Ordinateindicates percent of total NBD-PC flipped during 3 hours. Incubation wasin 2 mM Ca ²⁺, with correction for background measured in 0.1 mM EGTA.The error bars denote mean ± SD, n=7. Combined data of three independentexperiments performed on different days.

Because the conditions of incubation (neutral pH) were chosen to favorspecific cleavage of cysteinyl thioester bonds without disulfide bondreduction, these results imply an essential thioester linkage within theprotein. In a membrane-associated protein with cytoplasmic Cys residues,such as found in erythrocyte PL scramblase, this thiolester bond isnormally provided by palmitic acid in ester linkage to one or morecysteinyl thiols.(H. Schroeder, et al., J. Cell Biol. 134:647-660, 1996;M. Stauffenbiel, J. Biol. Chem. 263:13615-13622, 1988; C. A. Wilcox, etal., Biochemistry 26:1029-1036, 1987). Whereas the possibility ofdisulfide reduction by hydroxylamine cannot be excluded, it is importantto note that (1) virtually all cysteine residues in PL scramblase arenormally exposed to cytoplasmic reducing agents such as glutathione, anddisulfide bonds formation is therefore not anticipated and (2) Theabsence of any functionally-important disulfide bonds in PL scramblasecan be assumed based on the retention of normal PL scramblase activitywhen the protein was incubated in various reducing agents, includingdithiothreitol (F. Bassè, et al., supra, 1996; J. G. Stout, et al.,supra, 1997). Thus these data suggest that PL scramblase polypeptiderequires post-translational modification through addition of athiolester-linked fatty acid for its normal function in the plasmamembrane. Furthermore, these data imply that reagents that eitherprevent cellular acylation of the polypeptide, or, reagents that cleavecysteinyl thiolester linkages, will effectively inhibit endogenous PLscramblase activity.

9 1445 base pairs nucleic acid double linear cDNA unknown 1 CGCGGCCGCGTCGACCGAAA CCAGGAGCCG CGGGTGTTGG CGCAAAGGTT ACTCCCAGAC 60 CCTTTTCCGGCTGACTTCTG AGAAGGTTGC GCAGCAGCTG TGCCCGACAG TCTAGAGGCG 120 CAGAAGAGGAAGCCATCGCC TGGCCCCGGC TCTCTGGACC TTGTCTCGCT CGGGAGCGGA 180 AACAGCGGCAGCCAGAGAAC TGTTTTAATC ATGGACAAAC AAAACTCACA GATGAATGCT 240 TCTCACCCGGAAACAAACTT GCCAGTTGGG TATCCTCCTC AGTATCCACC GACAGCATTC 300 CAAGGACCTCCAGGATATAG TGGCTACCCT GGGCCCCAGG TCAGCTACCC ACCCCCACCA 360 GCCGGCCATTCAGGTCCTGG CCCAGCTGGC TTTCCTGTCC CAAATCAGCC AGTGTATAAT 420 CAGCCAGTATATAATCAGCC AGTTGGAGCT GCAGGGGTAC CATGGATGCC AGCGCCACAG 480 CCTCCATTAAACTGTCCACC TGGATTAGAA TATTTAAGTC AGATAGATCA GATACTGATT 540 CATCAGCAAATTGAACTTCT GGAAGTTTTA ACAGGTTTTG AAACTAATAA CAAATATGAA 600 ATTAAGAACAGCTTTGGACA GAGGGTTTAC TTTGCAGCGG AAGATACTGA TTGCTGTACC 660 CGAAATTGCTGTGGGCCATC TAGACCTTTT ACCTTGAGGA TTATTGATAA TATGGGTCAA 720 GAAGTCATAACTCTGGAGAG ACCACTAAGA TGTAGCAGCT GTTGTTGTCC CTGCTGCCTT 780 CAGGAGATAGAAATCCAAGC TCCTCCTGGT GTACCAATAG GTTATGTTAT TCAGACTTGG 840 CACCCATGTCTACCAAAGTT TACAATTCAA AATGAGAAAA GAGAGGATGT ACTAAAAATA 900 AGTGGTCCATGTGTTGTGTG CAGCTGTTGT GGAGATGTTG ATTTTGAGAT TAAATCTCTT 960 GATGAACAGTGTGTGGTTGG CAAAATTTCC AAGCACTGGA CTGGAATTTT GAGAGAGGCA 1020 TTTACAGACGCTGATAACTT TGGAATCCAG TTCCCTTTAG ACCTTGATGT TAAAATGAAA 1080 GCTGTAATGATTGGTGCCTG TTTCCTCATT GACTTCATGT TTTTTGAAAG CACTGGCAGC 1140 CAGGAACAAAAATCAGGAGT GTGGTAGTGG ATTAGTGAAA GTCTCCTCAG GAAATCTGAA 1200 GTCTGTATATTGATTGAGAC TATCTAAACT CATACCTGTA TGAATTAAGC TGTAAGGCCT 1260 GTAGCTCTGGTTGTATACTT TTGCTTTTCA AATTATAGTT TATCTTCTGT ATAACTGATT 1320 TATAAAGGTTTTTGTACATT TTTTAATACT CATTGTCAAT TTGAGAAAAA GGACATATGA 1380 GTTTTTGCATTTATTAATGA AACTTCCTTT GAAAAACTGC TTTAAAAAAA AGTCGACGCG 1440 GCCGC 1445318 amino acids amino acid double linear protein unknown 2 Met Asp LysGln Asn Ser Gln Met Asn Ala Ser His Pro Glu Thr Asn 1 5 10 15 Leu ProVal Gly Tyr Pro Pro Gln Tyr Pro Pro Thr Ala Phe Gln Gly 20 25 30 Pro ProGly Tyr Ser Gly Tyr Pro Gly Pro Gln Val Ser Tyr Pro Pro 35 40 45 Pro ProAla Gly His Ser Gly Pro Gly Pro Ala Gly Phe Pro Val Pro 50 55 60 Asn GlnPro Val Tyr Asn Gln Pro Val Tyr Asn Gln Pro Val Gly Ala 65 70 75 80 AlaGly Val Pro Trp Met Pro Ala Pro Gln Pro Pro Leu Asn Cys Pro 85 90 95 ProGly Leu Glu Tyr Leu Ser Gln Ile Asp Gln Ile Leu Ile His Gln 100 105 110Gln Ile Glu Leu Leu Glu Val Leu Thr Gly Phe Glu Thr Asn Asn Lys 115 120125 Tyr Glu Ile Lys Asn Ser Phe Gly Gln Arg Val Tyr Phe Ala Ala Glu 130135 140 Asp Thr Asp Cys Cys Thr Arg Asn Cys Cys Gly Pro Ser Arg Pro Phe145 150 155 160 Thr Leu Arg Ile Ile Asp Asn Met Gly Gln Glu Val Ile ThrLeu Glu 165 170 175 Arg Pro Leu Arg Cys Ser Ser Cys Cys Cys Pro Cys CysLeu Gln Glu 180 185 190 Ile Glu Ile Gln Ala Pro Pro Gly Val Pro Ile GlyTyr Val Ile Gln 195 200 205 Thr Trp His Pro Cys Leu Pro Lys Phe Thr IleGln Asn Glu Lys Arg 210 215 220 Glu Asp Val Leu Lys Ile Ser Gly Pro CysVal Val Cys Ser Cys Cys 225 230 235 240 Gly Asp Val Asp Phe Glu Ile LysSer Leu Asp Glu Gln Cys Val Val 245 250 255 Gly Lys Ile Ser Lys His TrpThr Gly Ile Leu Arg Glu Ala Phe Thr 260 265 270 Asp Ala Asp Asn Phe GlyIle Gln Phe Pro Leu Asp Leu Asp Val Lys 275 280 285 Met Lys Ala Val MetIle Gly Ala Cys Phe Leu Ile Asp Phe Met Phe 290 295 300 Phe Glu Ser ThrGly Ser Gln Glu Gln Lys Ser Gly Val Trp 305 310 315 1622 base pairsnucleic acid double linear cDNA unknown 3 TCTAAAGACT CAGGAAACAAAACCTAAATT GCCTCAAAGT TCAGGTGCTT TTTCTCCCTG 60 ACTTTAGTCT AGTGGAGTAGTGCAGCACCT ATGCCTTTCT GAGAGGAGTC TGGAGAGCTG 120 AGTCGCTGCT GGTGCTAGGATTCTAGGAAT TCGCCTCACT TGGAGCTGCA TGAGAAAAGA 180 AAGGCTTGCA AATGGAGGCTCCTCGCTCAG GAACATACTT GCCAGCTGGG TATGCCCCTC 240 AGTATCCTCC AGCAGCAGTCCAAGGACCTC CAGAGCATAC TGGACGCCCC ACATTCCAGA 300 CTAACTACCA AGTTCCCCAGTCTGGTTATC CAGGACCTCA GGCTAGCTAC ACAGTCTCAA 360 CATCTGGACA TGAAGGTTATGCTGCTACAC GGCTTCCTAT TCAAAATAAT CAGACTATAG 420 TCCTTGCAAA CACTCAGTGGATGCCAGCAC CACCACCTAT TCTGAACTGC CCACCTGGGC 480 TAGAATACTT AAATCAGATAGATCAGCTTC TGATTCATCA GCAAGTTGAA CTTCTAGAAG 540 TCTTAACAGG CTTTGAAACAAATAACAAAT TTGAAATCAA GAACAGCCTC GGGCAGATGG 600 TTTATGTTGC AGTGGAAGATACTGACTGCT GTACTCGAAA TTGCTGTGAA GCGTCTAGAC 660 CTTTCACCTT AAGAATCCTGGATCATCTGG GCCAAGAAGT CATGACTCTG GAGCGACCTC 720 TGAGATGCAG TAGCTGCTGCTTCCCCTGCT GCCTCCAGGA GATAGAAATC CAGGCTCCTC 780 CGGGGGTGCC AATAGGTTATGTGACTCAGA CCTGGCACCC ATGTCTGCCA AAGCTCACTC 840 TTCAGAACGA CAAGAGGGAGAATGTTCTAA AAGTAGTTGG TCCATGTGTT GCATGCACCT 900 GCTGTTCAGA TATTGACTTTGAGATCAAGT CTCTTGATGA AGTGACTAGA ATTGGTAAGA 960 TCACCAAGCA GTGGTCTGGTTGTGTGAAAG AGGCCTTCAC GGATTCGGAT AACTTTGGGA 1020 TCCAATTCCC GCTAGACCTGGAGGTGAAGA TGAAAGCTGT GACGCTTGGT GCTTGCTTCC 1080 TCATAGATTA CATGTTTTTTGAAGGCTGTG AGTAGGAACA GAAATCCGAC CTGCAGTAGG 1140 AATCAATGAA AGAGGACAGAGAAGATCTGA AGTCTACACA AGGAGATCAT ATGATTGAGA 1200 GACCTGGGGC TTTTTGATTTCTTCATTGAA ATTTCTCAGA ATCAAGCTGT TATACATGAA 1260 GCATAGTATG TAACATTTTGGTTTTCAAAT GGTAGTTTAT CTTTTACATT ATTGGAATAG 1320 ACCTGGATAA TTATCTTTATACACTTCTAA AAATATGCAC CAAATTCAAG TTAAAAAAAA 1380 AAAGACGAAG AGAAGTGTATGTTTTAAAAT AAAACATTTT ATGGAAAAGT AAGTTAAATC 1440 ATAATCTGGG ATTTATTTTTCATCTTTTGT TCAATTTAAA CCTTGTTAGT GCTGATTTTA 1500 TTATAAAATT GTACTTTACTATCAAACCTA GTTAGTTTAT TTCTTACAGA AATCCTCCTA 1560 TTATTTTGAA ATTACATATTTTTGAAAGCT TTTTAAAAGA TACTATTGCC TGGGAAATTC 1620 TA 1622 307 amino acidsamino acid double linear protein unknown 4 Met Glu Ala Pro Arg Ser GlyThr Tyr Leu Pro Ala Gly Tyr Ala Pro 1 5 10 15 Gln Tyr Pro Pro Ala AlaVal Gln Gly Pro Pro Glu His Thr Gly Arg 20 25 30 Pro Thr Phe Gln Thr AsnTyr Gln Val Pro Gln Ser Gly Tyr Pro Gly 35 40 45 Pro Gln Ala Ser Tyr ThrVal Ser Thr Ser Gly His Glu Gly Tyr Ala 50 55 60 Ala Thr Arg Leu Pro IleGln Asn Asn Gln Thr Ile Val Leu Ala Asn 65 70 75 80 Thr Gln Trp Met ProAla Pro Pro Pro Ile Leu Asn Cys Pro Pro Gly 85 90 95 Leu Glu Tyr Leu AsnGln Ile Asp Gln Leu Leu Ile His Gln Gln Val 100 105 110 Glu Leu Leu GluVal Leu Thr Gly Phe Glu Thr Asn Asn Lys Phe Glu 115 120 125 Ile Lys AsnSer Leu Gly Gln Met Val Tyr Val Ala Val Glu Asp Thr 130 135 140 Asp CysCys Thr Arg Asn Cys Cys Glu Ala Ser Arg Pro Phe Thr Leu 145 150 155 160Arg Ile Leu Asp His Leu Gly Gln Glu Val Met Thr Leu Glu Arg Pro 165 170175 Leu Arg Cys Ser Ser Cys Cys Phe Pro Cys Cys Leu Gln Glu Ile Glu 180185 190 Ile Gln Ala Pro Pro Gly Val Pro Ile Gly Tyr Val Thr Gln Thr Trp195 200 205 His Pro Cys Leu Pro Lys Leu Thr Leu Gln Asn Asp Lys Arg GluAsn 210 215 220 Val Leu Lys Val Val Gly Pro Cys Val Ala Cys Thr Cys CysSer Asp 225 230 235 240 Ile Asp Phe Glu Ile Lys Ser Leu Asp Glu Val ThrArg Ile Gly Lys 245 250 255 Ile Thr Lys Gln Trp Ser Gly Cys Val Lys GluAla Phe Thr Asp Ser 260 265 270 Asp Asn Phe Gly Ile Gln Phe Pro Leu AspLeu Glu Val Lys Met Lys 275 280 285 Ala Val Thr Leu Gly Ala Cys Phe LeuIle Asp Tyr Met Phe Phe Glu 290 295 300 Gly Cys Glu 305 14 amino acidsamino acid single linear peptide unknown 5 Cys Glu Ser Thr Gly Ser GlnGlu Gln Lys Ser Gly Val Trp 1 5 10 39 base pairs nucleic acid singlelinear oligonucleotide unknown 6 TCAGAATTCG GATCCATGGA CAAACAAAACTCACAGATG 39 43 base pairs nucleic acid single linear oligonucleotideunknown 7 GCTTGCCTGC AGGTCGACCT ACCACACTCC TGATTTTTGT TCC 43 38 basepairs nucleic acid single linear oligonucleotide unknown 8 TCAGAATTCGGATCCATGGA GGCTCCTCGC TCAGGAAC 38 43 base pairs nucleic acid singlelinear oligonucleotide unknown 9 GCTTGCCTGC AGGTCGACCT ACACACAGCCTTCAAAAAAC ATG 43

We claim:
 1. An isolated preparation of phospholipid scramblase, whereinthe protein is approximately 35 Kd as measured on a 12.5%SDS-polyacrylamide gel under reducing conditions, wherein the scramblesis a naturally occuring phospholipids scramblase.
 2. The preparation ofclaim 1 wherein the protein comprises SEQ ID NO:2.
 3. The preparation ofclaim 1 wherein the protein comprises amino acid residues 85-309 of SEQID NO:2.
 4. The preparation of claim 1 wherein the scramblase is anaturally occuring mouse phospholipid scramblase.
 5. The preparation ofclaim 1, wherein the phospholipid scramblase comprises SEQ ID NO:4. 6.The preparation of claim 1, wherein the phospholipid scramblasecomprises amino acid residues 83-307 of SEQ ID NO:4.
 7. The preparationof claim 4 wherein the protein is isolated from mouse erythrocytemembranes.
 8. A recombinant DNA sequence encoding PL scramblase, whereinthe sequence is SEQ ID NO:1.
 9. An isolated DNA sequence comprisingnucleotides 211-1164 of SEQ ID NO:1.
 10. An isolated DNA sequencecomprising nucleotides 463-1137 sequence ID NO:1.
 11. An isolated DNAsequence wherein the sequence is SEQ ID NO:3.
 12. An isolated DNAsequence comprising nucleotides 192-1112 of SEQ ID NO:3.
 13. An isolatedDNA sequence comprising nucleotides 438-112 of SEQ ID NO:3.
 14. Anisolated DNA sequence wherein the sequence is SEQ ID NO:1 and whereinthe sequence is part of a protein expression vector.
 15. An isolated DNAsequence comprising nucleotides 211-1164 of SEQ ID NO:1 and wherein thesequence is part of a protein expression vector.
 16. An isolated DNAsequence comprising nucleotides 463-1137 of SEQ ID NO:1 and wherein thesequence is part of a protein expression vector.
 17. An isolated DNAsequence wherein the sequence is SEQ ID NO:3 and wherein the sequence ispart of a protein expression vector.
 18. An isolated DNA sequencewherein the sequence comprises 192-1112 of SEQ ID NO:3 and wherein thesequence is part of a protein expression vector.
 19. An isolated DNAsequence comprising nucleotides 438-1112 of SEQ ID NO:3 and wherein thesequence is part of a protein expression vector.
 20. A recombinant DNAsequence encoding PL scramblase, wherein the sequence has been modifiedto prevent post-translational modification in the encoded PL scramblaseand wherein the sequence comprises a mutation with a non-functionalequivalent substitution of residue Thr161 of SEQ ID NO:2; Thr159 of SEQID NO:4 or the equivalent residue in the conserved region of another PLscramblase.
 21. A recombinant DNA sequence encoding PL scramblase,wherein the sequence has been modified to prevent post-translationalmodification in the encoded PL scramblase and wherein the sequencecomprises at least one non-functional equivalent substitution withinresidues Asp273-Asp284 of SEQ ID NO:2; Asp271-Asp282 of SEQ ID NO:4 orthe equivalent residue in the conserved region of another PL scramblase.22. A recombinant DNA sequence encoding PL scramblase, wherein thesequence has been modified to prevent post-translational modification inthe encoded PL scramblase and wherein the sequence comprises a mutationat Cys297 of SEQ ID NO:2 or the equivalent residue in the conservedregion of another PL scramblase.
 23. An isolated protein encoded by thesequence of claim
 20. 24. An isolated protein encoded by the sequence ofclaim
 21. 25. An isolated protein encoded by the sequence of claim 22.26. A method of inhibiting expression of the coagulant properties of theplasma membrane of a cell comprising the step of expressing in the cellplasma membrane a mutant PL scramblase, wherein the PL scramblase has areduced activity in mediating transmembrane movement of plasma membranephospholipids, and wherein the mutant phospholipid scramblase comprisesa non-functionally equivalent substitution at residue Thr161 of SEQ IDNO:2; Thr159 of SEQ ID NO:4 or the equivalent residue in the conservedregion of-another PL scramblase.
 27. A method of inhibiting expressionof the coagulant properties of the plasma membrane of a cell comprisingthe step of delivering in the cell plasma membrane a mutant PLscramblase, wherein the PL scramblase has a reduced activity inmediating transmembrane movement of plasma membrane phospholipids, andwherein the mutant phospholipid scramblase comprises a non-functionallyequivalent substitution of a cysteine residue wherein the cysteineresidue to be substituted is Cys297 of SEQ ID NO:2 or Cys295 of SEQ IDNO:4.
 28. A method of inhibiting expression of the coagulant propertiesof the plasma membrane of a cell comprising the step of delivering inthe cell plasma membrane a mutant PL scramblase, wherein the PLscramblase has a reduced activity in mediating transmembrane movement ofplasma membrane phospholipids, and wherein the mutant phospholipidscramblase comprises a non-functionally equivalent substitution of atleast one of the residues located in the region of Asp273-Asp284 of SEQID NO:2;, Asp271-Asp282 of SEQ ID NO:4 or the equivalent residues in theconserved region of another PL scramblase.
 29. A method of inhibitingexpression of the coagulant properties of the plasma membrane of a cellcomprising the step of delivering in the cell plasma membrane a mutantPL scramblase, wherein the PL scramblase has a reduced activity inmediating transmembrane movement of plasma membrane phospholipids, andwherein the mutant phospholipid scramblase comprises a non-functionallyequivalent substitution of at least one residue selected from the groupconsisting of Asp273, Asp275, Phe277, Ile279, Phe281, and Asp284 of SEQID NO:2; and Asp271, Asp273, Phe275, Ile277, Phe279, and Asp282 of SEQID NO:4.